Apparatus for high throughput chemical reactions

ABSTRACT

Apparatus, systems, chips, and methods of performing a large number of simultaneous chemical reactions are provided herein. The chips of the invention comprise addressable units that can be addressed according to the temperature of the reaction to be run. The subject apparatus, systems, and chips are particularly suited for performing polymerase chain reactions on thousands of nucleic acid sequences, up to and including sequences of an entire genome of an organism of interest.

CROSS-REFERENCE

This application claims the benefit of U.S. Provisional Application No.60/886,067, filed Jan. 22, 2007 and U.S. Provisional Application No.61/016,377, filed Dec. 21, 2007, which applications are incorporatedherein by reference in their entirety.

BACKGROUND OF THE INVENTION

The advent and development of Polymerase Chain Reaction (PCR) since 1983has revolutionized molecular biology through vastly extending thecapability to identify, manipulate, and reproduce DNA. A number ofdifferent applications have been developed to utilize PCR, such asscientific research, clinical diagnostics, forensic identifications, andenvironmental studies.

Following the sequencing of the human genome, genomic analysis of theestimated 30,000 human genes has been a major focus of basic and appliedbiochemical and pharmaceutical research. Diagnostics, medicines, andtherapies for a variety of disorders may be developed from the analysisand manipulation of genes. Diagnostic devices often utilize smallsamples from patients. Patient samples collected for diagnostic purposesare typically of limited quantity and volume and thus only a smallnumber of tests can be performed on a single sample. Therefore, there isneed for a miniaturized device capable of performing analysis of a largenumber of genes or nucleic acid sequences from a single small sample.

Development of gene-based therapies has also become a major focus forboth researchers and pharmaceuticals. In order to develop new therapiesand recognize new therapeutic targets, high-throughput screeningutilizing most, if not all, of an entire genome of an organism would bedesirable. In addition, the ability to sequence and amplify an entiregenome from a sample from an individual may pave the way for thedevelopment of personal medicines.

Many of the PCR microplates and thermocyclers currently available areunable to performing a large quantity of PCR at a reasonable cost. Inmany reactions, the sample volume needed to analyze each individualsequence is on the order of microliters. When sequencing or amplifyingthousands of genes, the amount of sample needed from an individual orgroup of individuals often becomes not practical. In addition, whendealing with a large number of sequences, the sensitivity andspecificity of the reactions become a major issue when performing PCR.The annealing temperatures necessary for PCR amplification of a sequencecan vary by as much as 15° C. from sequence to sequence. In order tosequence thousands of genes from a relatively small sample, a thermalcycling apparatus needs to adapt to range of different temperatures.

In recent years, the advancement in nanofabrication technology enabledthe production of miniaturized devices integrated with electrical,optical, chemical or mechanical elements. The technology embodies arange of fabrication techniques including low-pressure vapor deposition,photolithography, and etching. Based on these techniques, miniaturizeddevices containing silicon channels coupled to nano-heaters have beenproposed (see, for example, U.S. Pat. Nos. 6,962,821, 6,054,263,5,779,981 and 5,525,300). While the channel- or chamber-based design inprinciple reduces the thermal mass and the reaction volume, it stillsuffers from other practical drawbacks. In particular, the channels orchambers by design are limited with respect to controlling temperatureand evaporation.

Such devices or systems would greatly aid in diagnostic testing,pharmaceutical development, and personal medicine. The present inventionsatisfies this need and provides related advantages as well.

SUMMARY OF THE INVENTION

In general in one aspect an apparatus is provided comprising at leastone heating element, configured to be in thermal contact with a chipsaid chip comprising a substrate and an array of nanowells, wherein theat least one heating element is configured to move relative to the chip.

In one embodiment heating element is in thermal contact with the chipfrom above and below the chip, and wherein the heating element inthermal contact from below the chip is set at a temperature lower thanthe temperature of the heating element in thermal contact from above thechip. In another embodiment the chip comprises an upper surface and abottom surface and wherein a first series of nanowells is arranged alongone orientation on the upper surface and a second series of nanowells isoriented perpendicular to the first series of nanowells. The heatingelement can be positioned above or below a stationary chip comprising anarray of nanowells and the heating element can be capable of heating andcooling.

In one embodiment the apparatus includes a plurality of heating elementscorresponding to a plurality of temperature zones. The plurality oftemperature zones can be within a range from about 52° C. to about 95°C. In another embodiment the plurality of temperature zones provides atemperature gradient. The at least one of the temperature zones can beset at a temperature ranging from about 52° C. to about 65° C. and atleast one other temperature zone can be set at a temperature rangingfrom about 90° C. to about 95° C. In a further embodiment at least oneother temperature zone set at an elongation temperature ranging fromabout 68° C. to about 72° C.

In a particular embodiment the at least one heating element isconfigured to provide an output comprising a spike waveform oftemperature over time.

In one embodiment an individual nanowell in said array has a dimensionof about 250 μm in length, about 250 μm in width, and a depth of about525 μm, or less.

In another embodiment the chip is operatively coupled to an opticalsystem that detects optical signals. The optical system can comprise aplurality of optical detectors.

In one embodiment the number of nanowells is greater than about 30,000.In another embodiment the nanowells are configured to contain about 100nl.

In general in another aspect a method of conducting a chemical reactionis provided comprising providing a chip configured to receive a reactionsample; providing at least one heating element positionable in relationto the chip to provide thermal contact between the at least one heatingelement and the chip; and conducting the chemical reaction in thereaction sample by varying the temperature of the chip, wherein saidvarying the temperature is effected by moving the at least one heatingelement in relation to the chip such that the heating element is inthermal contact with the chip.

In one embodiment the chemical reaction is a nucleic acid amplificationreaction.

In another embodiment movement of the at least one heating element iscontrolled by signals generated from a temperature sensor that isoperatively linked to the chip.

In a further embodiment the reaction sample is capable of producing anoptical signal, and, wherein the chip is operatively coupled to anoptical system configured to detect optical signals emitted from thereaction sample. The optical signals can be proportional to the amountof product of the chemical reaction.

In one embodiment varying the temperature is effected by moving aplurality of heating elements, each of which is set at a differenttemperature. In another embodiment at least one heating element is setat a temperature ranging from about 52° C. to about 65° C. and at leastone other heating element is set at a temperature ranging from about 90°C. to about 95° C.

In general in another aspect a chip for running a reaction is providedcomprising an array of addressable units, each unit being configured fora chemical reaction, wherein the array of the addressable units isconfigured to correspond to a predetermined temperature zone, andwherein an individual unit in said array is dimensioned to hold achemical reaction mixture of less than about 1 μl. In one embodiment theapparatus is comprising a plurality of arrays. In another embodiment theapparatus includes a plurality of arrays, each of which corresponding toa different temperature zone. In one embodiment at least one of thearrays is set at an annealing temperature for supporting a nucleic acidamplification reaction and at least one other array is set at adenaturing temperature for supporting a nucleic acid amplificationreaction.

In a particular embodiment the zone is addressed to indicate thepredetermined temperature zones. In another embodiment the array ofaddressable units are configured to correspond to six or morepredetermined temperature zones.

In one embodiment the chip is in thermal contact with a heating element.

In general in another aspect an apparatus is provided for conducting achemical reaction requiring cycling at least two temperature levels,comprising: (a) chip for running a reaction comprising an array ofaddressable units, each unit being configured for a chemical reaction,wherein the array of the addressable units is configured to correspondto a predetermined temperature zone, and wherein an individual unit insaid array is dimensioned to hold a chemical reaction mixture of lessthan about 1 μl; and (b) a heating element in thermal contact with thechip.

In one embodiment the array of addressable units is greater than about30,000.

In a particular embodiment the apparatus is further comprising (c) anoptical system operatively coupled to the chip, wherein the opticalsystem detects an optical signal coming from an addressedthermo-controllable unit. In one embodiment the optical system comprisesa plurality of optical detectors.

In one embodiment the apparatus is further comprising a plurality ofheating elements. In a particular embodiment the plurality of heatingelements comprises six or more heating elements. In one embodiment anindividual unit within the array comprises a nanowell for receiving andconfining a sample, said well being sealed when filled with the sample.In another embodiment the chemical reaction is a nucleic acidamplification reaction. In one embodiment the predetermined temperatureof a unit is configured to yield at least 90% of homogeneous productfrom the chemical reaction.

In general in another aspect a method of conducting a reaction thatinvolves a plurality of reaction samples and requires cycling at leasttwo temperature levels is provided comprising: (a) providing a chip forrunning a reaction comprising an array of addressable units, each unitbeing configured for a chemical reaction, wherein the array of theaddressable units is configured to correspond to a predeterminedtemperature zone, and wherein an individual unit in said array isdimensioned to hold a chemical reaction mixture of less than about 1 μl;(b) placing the plurality of reaction samples into the units of the chipaccording to the set of predetermined temperatures; and (c) controllinga heating element to effect cycling at least two temperature levels.

In one embodiment an individual unit within the array of the chipcomprises a nanowell for receiving and confining a sample, said wellbeing sealed when filled with the sample. In another embodiment thechemical reaction is a nucleic acid amplification reaction. In a furtherembodiment the predetermined temperature of a unit is configured toyield at least 90% of homogeneous product from the chemical reaction.

In general in yet another aspect an apparatus for conducting a chemicalreaction involving cycling at least two temperature levels is providedcomprising: (a) a body configured to receive a chip comprising aplurality of nanowells for containing the chemical reaction; and (b) afirst heater providing a first temperature and a second heater providinga second temperature; wherein the first heater and the second heater areconfigured to be movable between a first and a second orientation, andwherein the first orientation places the heater in thermal contact withthe sample holder and the second orientation does not place the heaterin thermal contact with the sample holder.

In one embodiment the plurality of nanowells are addressable, whereinthe nanowells are arranged according to a predetermined set oftemperatures, such that at least one of the nanowells is addressed toindicate the predetermined temperature for running the chemical reactionwithin said nanowell. In a particular embodiment the plurality ofnanowells comprises over about 30,000 nanowells.

In one embodiment the first heater comprises a plurality of temperaturezones. In another embodiment the temperature zones comprise six or moretemperature zones. In a further embodiment the plurality of temperaturezones correspond to the predetermined set of temperatures according towhich the thermo-controllable units are arrayed. In one embodiment thefirst and second heaters move between the first and second orientationsaccording to a protocol.

In a further embodiment the apparatus is comprising a motor for removingthe first and second heaters between the first and second orientations.

In one embodiment the first heater can provide a temperature gradient.In another embodiment the apparatus is further comprising a heat sink inthermal contact with the first heater. In a different embodiment theapparatus is further comprising a heat sink in thermal contact with thesecond heater.

In some embodiments the apparatus is further comprising a fan forremoving heat from the heat sink. In other embodiments the apparatus isfurther comprising a plurality of temperature sensors operably connectedto the chip. In one embodiment the plurality of temperature sensors hasat least one temperature sensor assigned to measure the temperature ofeach temperature zone.

INCORPORATION BY REFERENCE

All publications and patent applications mentioned in this specificationare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIG. 1 illustrates an exemplary embodiment of a chip of the inventionthat comprises six smaller chips or six arrays of addressable unitsrepresenting different addressed predetermined temperatures.

FIG. 2 is a top view of one exemplary chip layout on a circularsubstrate.

FIG. 3 is a top view of one exemplary chip layout showing nanowells onthe chip.

FIG. 4 is a longitudinal cross section of the side view of an exemplarynanowell chip.

FIG. 5 illustrates an exemplary chip of the invention comprising a setof smaller chips that each represents a different temperature zone.

FIG. 6 depicts one illustrative apparatus design having at least oneheating element with a nanowell chip.

FIG. 7 is an illustrative drawing of an apparatus of one embodiment ofthe invention with more than one temperature zone.

FIG. 8 is a side view of one embodiment of apparatus of one embodimentof the invention with a top mounted heating element and an opticalscanner.

FIG. 9 is a graph plotting the changes in temperature of apparatus(y-axis) at various times (x-axis).

FIG. 10 depicts a series of thermal temperature profiles capable ofbeing produced by the arc lamps. Each temperature profile shows the chipresponse to the heating element. The top row of graphs (a) shows thechange in temperature (y-axis) over time (x-axis). The bottom row ofgraphs (b) show the temperature of the chip (y-axis) over the depth ofthe chip (x-axis).

FIGS. 11 a-c represent examples of different heating elementconfigurations of a thermal cycling apparatus.

FIG. 12 demonstrates an exemplary embodiment of a thermal cyclingapparatus wherein two heaters are movable between a first orientationand second orientation, wherein the first orientation is in thermalcontact with a sample holder.

FIG. 13 demonstrates a side view of the first heater from the example inFIG. 12 when the heater is in the first orientation in thermal contactwith a chip with addressable units.

FIG. 14 demonstrates a temperature profile provided by a thermal cyclingapparatus of the invention with a heater divided into differenttemperature zones.

FIG. 15 demonstrates an exemplary apparatus or system of the invention,wherein the apparatus comprises a top cover slide and a heater capableof providing force to bring a chip or chemical reaction into opticaland/or thermal contact with the top cover slide.

FIG. 16 is a schematic drawing of one embodiment of the thermocyclingsystem of the invention.

FIG. 17 illustrates an exemplary system of the invention comprising achip, a heating apparatus, and an optical system for analyzing thereaction results.

FIG. 18 demonstrates an example system of the invention comprising anoptical system, a heating apparatus, and a chip for conducting achemical reaction.

FIG. 19 is a block diagram showing a representative example logic devicein communication with the system according to the specific embodimentsof the invention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides chips, thermal cycling apparatuses,systems, and methods for conducting a plurality of chemical reactionsand for multiplexed analyses of individual molecules. The presentinvention also provides miniaturized, highly automated devices andmethods that allow the manipulation of the precise control of thereaction substance, conditions and temperature.

The present invention can relate to methods, chips, and apparatuses forsimultaneously analyzing a whole genome of an organism. Many of themethods relate to the qualitative and/or quantitative analysis of agenomic mixture of nucleotides, using polymerase chain reaction orsimilar amplification methods conducted in very small reaction volumes.

The analysis of the estimated 30,000 human genes may provide methods forapplied pharmaceutical research and developing diagnostics, medicinesand therapies for wide variety of disorders. For example, throughunderstanding of genetic differences between normal and diseasedindividuals, differences in the biochemical makeup and function of cellsand tissues can be determined and appropriate therapeutic interventionsidentified.

In an embodiment, the genome may be from humans, mammals, mice,Arabidopsis or any other plant, bacteria, fungi or animal species. Theinvention may be used for drug discovery and for diagnostics of aparticular individual, animal or plant.

In many situations, it can be desirable to determine the gene expressionprofile from a test of all genes in an organism. Such a test can also beuseful to screen DNA or RNA from a single individual for sequencevariants associated with different mutations in the same or differentgenes (for example, single nucleotide polymorphisms, or “SNPs”), or forsequence variants that serve as markers for the inheritance of differentchromosomal segments from a parent. Such tests can also be useful, forexample, to predict susceptibility to disease, determine whether anindividual is a carrier of a genetic mutation, determine whether anindividual may be susceptible to adverse reactions or resistance tocertain drugs, or for other diagnostic, therapeutic or researchpurposes.

Chips

The overall size of a chip of the invention may vary and it can rangefrom a few microns to a few centimeters in thickness, and from a fewmillimeters to 50 centimeters in width or length. Typically, the size ofthe entire chip ranges from about 10 mm to about 200 mm in width and/orlength, and about 1 mm to about 10 mm in thickness. In some embodiments,the chip is about 40 mm in width by 40 mm in length by 3 mm inthickness.

The chip can also be a set of smaller chips. For example, the chip cancomprise six smaller chips (e.g., six arrays of addressable units) witha thermal buffer between each of the smaller chips. A chip that is a setof smaller chips is also referred to herein as a plate. In an embodimentof the example, each of the six smaller chips corresponds to a differentpredetermined temperature to which the array of units in the overallchip are addressed.

The total number of units on the chip will vary depending on theparticular application in which the subject chips are to be employed.The density of the units on the chip surface may vary depending on theparticular application. The density of units (for example, number ofchambers per unit surface area of substrate), and the size and volume ofunits, may vary depending on the desired application and such factorsas, for example, the species of the organism for which the methods ofthis invention are to be employed.

A large number of units may be incorporated into a chip of theinvention. In various embodiments, the total number of units on the chipis from about 1000 to about 200,000, more preferably from about 5000 toabout 100,000. In other embodiments the chip comprises smaller chips,each of which comprises about 5,000 to about 20,000 units. Therefore, ifthe larger chip comprises about 3 to about 20 smaller chips, itcomprises about 15,000 to about 400,000 units. In certain embodiments,the chip comprises about 100,000 units.

For example, a square chip may comprise 125 by 125 nanowells, with adiameter of 0.1 mm. Table I shows some examples of the well layoutformat for certain exemplary chips of the invention. A plate isequivalent to a chip comprising multiple smaller chips as describedherein. In the example of Table I, a plate comprises six smaller chips.

TABLE I m n depth ~Wells/Chip Wells/plate (approx.) (approx.) (mm)(approx.) (approx.) 125 125 0.1 15743 94459 122 122 0.11 14790 88742 118118 0.12 13921 83527 115 115 0.13 13126 78756 111 111 0.14 12397 74381108 108 0.15 11726 70358 105 105 0.16 11108 66651 103 103 0.17 1053863228 100 100 0.18 10010 60060 98 98 0.19 9521 57124 95 95 0.2 906654396 93 93 0.21 8643 51858 91 91 0.22 8249 49492 89 89 0.23 7881 4728387 87 0.24 7536 45218 85 85 0.25 7214 43285 83 83 0.26 6912 41472

In Table I m is an approximate number of wells along a horizontal axis,n is an approximate number of wells along a perpendicular axis, depth ismeasured in mm, and the number of wells/chip and number of wells/plateare approximate.

The chip can be of any size or have any number of units. In anembodiment, a user or a customer receiving a chip of the inventionchooses the size, units and whether a unit comprises a nanowell. In apreferable embodiment, when running a nucleic acid amplificationchemical reaction with a unit on a chip of the invention, the user canselect the number of units according to the number of genes required tosequence an entire genome of a species.

An example chip 100 of the embodiment comprising six smaller chips 110is illustrated in FIG. 1. The smaller chips 110 are 38 mm by 39.5 mm inarea, and the overall size of the chip 100 comprising the six smallerchips 110 is about 85 mm by 129 mm in area. In this example, the smallerchips 110 comprise a large well size of about 400 μm (not represented inscale in the figure). The smaller chips 110 can comprise a series of mby n nanowells 120 as demonstrated in Table I. In this embodiment, thet-mm wall thickness 122 can determine the number of nanowells on a chip.Each of the smaller chips 110 can represent a predetermined temperatureof the overall chip 100 and thus, each smaller chip 110 can be addressedaccording to the temperature of the reaction to be run in each nanowell120. In addition, when a plurality of smaller chips 110 are used in anoverall larger chip 100, a thermally insulative frame 130 can thermallyisolate each addressable predetermined temperature area from oneanother. In this example, the thermally insulative frame 130 is 3 mm inthickness and can be made of mica, polyethylene, or any other insulativematerial as would be obvious to one skilled in the art.

In an embodiment, a chip can run 33,750 assays for whole genome, highthroughput gene expression real-time PCR.

The nanowell may be fabricated in any convenient size, shape or volume.The well may be about 100 μm to about 1 mm in length, about 100 μm toabout 1 mm in width, and about 100 μm to about 1 mm in depth. In variousembodiments, each nanowell has an aspect ratio (ratio of depth to width)of from about 1 to about 4. In one embodiment, each nanowell has anaspect ratio of about 2. The transverse sectional area may be circular,elliptical, oval, conical, rectangular, triangular, polyhedral, or inany other shape. The transverse area at any given depth of the well mayalso vary in size and shape.

In an embodiment, the nanowell can have a volume of from about 1 nl toabout 1 ul. The nanowell typically has a volume of less than 1 ul,preferably less than 500 nl. The volume may be less than 200 nl, or evenless than 100 nl. In an embodiment, the volume of the nanowell is about100 nl. Where desired, the nanowell can be fabricated to increase thesurface area to volume ratio, thereby facilitating heat transfer throughthe unit, which can reduce the ramp time of a thermal cycle.

The cavity of each nanowell may take a variety of configurations. Forinstance, the cavity within a nanowell may be divided by linear orcurved walls to form separate but adjacent compartments, or by circularwalls to form inner and outer annular compartments.

A nanowell of high inner surface to volume ratio may be coated withmaterials to reduce the possibility that the reactants contained thereinmay interact with the inner surfaces of the well. Coating isparticularly useful if the reagents are prone to interact or adhere tothe inner surfaces undesirably. Depending on the properties of thereactants, hydrophobic or hydrophilic coatings may be selected. Avariety of appropriate coating materials are available in the art. Someof the materials may covalently adhere to the surface, others may attachto the surface via non-covalent interactions. Non-limiting examples ofcoating materials include silanization reagent such asdimethychlorosilane, dimethydichlorosilane, hexamethyldisilazane ortrimethylchlorosilane, polymaleimide, and siliconizing reagents such assilicon oxide, Aquasil™, and Surfasil™. Additional suitable coatingmaterials are blocking agents such as amino acids, or polymers includingbut not limited to polyvinylpyrrolidone, polyadenylic acid andpolymaleimide.

Certain coating materials can be cross-linked to the surface viaextensive heating, radiation, and by chemical reactions. Those skilledin the art will know of other suitable means for coating a nanowell of achip, or will be able to ascertain such, without undue experimentation.

In an embodiment, an individual unit of the chip comprises a nanowellfor receiving and confining a sample, said well being sealed when filledwith the sample.

The individual units within the array can be separated from each otherby a physical barrier resistant to the passage of liquids. In oneaspect, these units may comprise indented areas referred to asnanowells. A nanowell can be open at the top, but is physically isolatedfrom other wells to restrict passage of liquids. Accordingly, thenanowell has at least one cavity suitable for receiving and confiningreaction sample. In order to isolate one nanowell from the environmentto restrict the passage of liquids, the nanowell can be sealed. In apreferable embodiment, a method of sealing a nanowell is depositingmineral oil on top of the sample within the well to confine the sample.The mineral oil can be nano-dispensed. A nanowell can be sealed by anymethod as would be obvious to those skilled in the art.

In many applications, sealing nanowells is desirable to preventevaporation of liquids and thus maintains the preferred reactionconcentrations throughout the thermal cycling. Accordingly, a techniquefor sealing an array of nanowells can be employed. A useful sealingtechnique takes several factors into consideration. First, the methodshould be amenable to high throughout processing of a large quantity ofnanowells. Second, the method should permit selective sealing ofindividual nanowells. As such, the method can yield chips comprisingopen nanowells interspersed among sealed nanowells in any desiredpattern or format. An open and/or unfilled well can not only allowpassive dissipation of heat, but also can reduce heat transfer betweenthe neighboring nanowells.

An alternative method of sealing results in an array of nanowellscontaining at least one open well. The method can include the steps of(a) applying a radiation-curable adhesive along peripheral dimensionsdefining the open surface of the at least one open nanowell; (b) placinga cover to encompass the peripheral dimensions that define the opensurface of the at least one open nanowell that is to be sealed; and (c)exposing the array to a radiation beam to effect the sealing.

As used herein, “radiation-curable adhesive” refers to any compositionthat cures and bonds to the adhering surface upon exposure to aradiation beam without the need of extensive heating. “Radiation beam”refers to electromagnetic waves of energy including, in an ascendingorder of frequency, infrared radiation, visible light, ultraviolet (UV)light, X-rays, and gamma rays. A vast number of radiation-curableadhesive are commercially available (see, for example, a list ofcompanies selling radiation-curable adhesive and radiation systems fromThomasNet®'s worldwide web site). Such materials include a diversity ofacrylics, acrylates, Polyurethanes (PUR), polyesters, vinyl, vinylesters, and a vast number of epoxies that are curable by radiation beamsat various frequencies. These and other radiation-curable materials aresupplied commercially in form of liquid, or solid such as paste, powder,resin, and tape.

The choice of radiation-curable adhesive will be dependent on thematerial make up of the surfaces to be adhered. The aforementionedclasses of adhesive are suited for adhering the chip substrate to thecover which can be made of a range of materials. For instance, acrylicsand epoxies are applicable for radiation-sealing any two surfaces, madeof any one of the materials selected from glass, ceramics, metalloids,semiconductors (for example, silicon, silicates, silicon nitride,silicon dioxide, quartz, and gallium arsenide), plastics, and otherorganic polymeric materials. Radiation-curable materials exhibiting theproperties of low use temperature and rapid curing time can be desirablefor sealing the subject chips. These materials allow for a rapid sealingto avoid radiation damages to the chemical or biological reagentscontained in the chips.

The radiation-curable adhesive can be applied by any mechanical meansalong the peripheral dimensions that define the open surface of ananowell. The “peripheral dimensions” can be the boundaries on the chipsubstrate or on the cover. In either case, the peripheral dimensionsbecome bonded to the respective adhering surface, the substrate or thecover, upon curing the adhesive. The radiation-curable adhesive can besmeared, printed, dispensed, or sprayed onto the peripheral dimensionsusing any suitable tools. Mechanical means can yield a uniform layer ofadhesive on the peripheral dimensions. One way to provide a uniformdistribution is to apply the adhesive directly onto the peripheraldimensions of an open well using a squeegee over a meshed screen mask.Alternatively, the radiation-curable adhesive can be applied directlyonto the cover that has been marked with the peripheral dimensions usingthe meshed screen mask. A uniform layer of adhesive is achieved uponremoval of the mask.

Upon application of the radiation-curable adhesive, a cover is placed onthe nanowell to encompass the peripheral dimensions that define the opensurface of the well. Suitable covers are generally made of materialsthat permit passage of a radiation beam. Preferred covers are fabricatedwith transparent materials such as glass quartz, plastic, any suitableorganic polymeric materials known to those skilled in the art, or anycombinations thereof.

Sealing a covered nanowell can be carried out by exposing the well to aradiation beam. Depending on the type of adhesive selected, theradiation beam may come from a conventional incandescent source, alaser, a laser diode, UV-bulb, an X-ray machine or gamma-ray machine, orthe like. Where desired, radiation beam from the radiation source ispermitted to reach only selected locations on the nanowell array so thatonly certain selected wells are to be sealed. A selective sealing isoften achieved by using a photo-mask patterned with the locations of thenanowells. The photo-mask is provided with transparent locations andopaque locations that correspond to the nanowells that are to be sealedand those that are to remain open, respectively. The radiation beampasses freely through the transparent regions but is reflected from orabsorbed by the opaque regions. Therefore, only selected nanowells areexposed to light and hence sealed by curing the adhesive. The photo-maskcan be patterned such that no two adjoining open nanowells are to besealed. The photo-mask can be patterned such that the resulting nanowellarray contains alternating sealed and unsealed wells. One skilled in theart can fashion an unlimited number of photo-masks with any patterns toyield chips containing open and sealed nanowells in any format. Methodsfor manufacturing such photo-masks are well established in the art andhence are not detailed herein.

FIG. 2 is a representative schematic drawing of an alternative chip 300made from a silicon wafer 200. Preferred silicon chips have an overallsize of about 500 microns in thickness and may have any width or lengthdepending on the number of nanowells desired. Such a silicon wafer 200is 6 inches (150 mm) in diameter and 626 μm thick. A chip 300 can befabricated from such a wafer 200, such chip being approximately 85.48 mmalong one side 210, 127.76 mm along the other side 220, and 150 mm alongthe diagonal length of the chip 250. As fabricated, the chip is an SBScompliant qPCR chip. The total number of nanowells fabricated on thechip will vary depending on the particular application in which thesubject chips are to be employed. To accommodate the need forsimultaneous performance of a vast number of reactions, the subjectchips will generally comprise at least 100 nanowells, and more usuallyover 30,000 nanowells. The density of the nanowells on the chip surfacemay vary depending on the particular application. For example, thedensity of nanowells on the chip surface can range between about 1 toabout 1000 nanowells per mm². In another example the density ofnanowells on the chip can range between about 10 to about 100 nanowellsper mm².

FIG. 3 is an illustrative diagram of the top side of a chip 300 withrepresentative nanowells 302. In one exemplary embodiment, the nanowells302 of the chip 300 are 0.25 mm in length and 0.25 mm in width and thecenters of the nanowells 302 are spaced 0.348 mm apart. It is envisionedthat the centers of the nanowells 302 can be spaced as desired,including, for example between 2 mm and 0.01 mm apart. The nanowells ofthe subject chips can be arrayed in any format across or over thesurface of the chip, such as in rows and columns so as to form a grid,in a particular pattern, and the like as seen in FIG. 3. In a preferredembodiment, the nanowells are arrayed in a format compatible toinstrumentation already existing for dispensing reagents and/or readingassays, such that engineering of commercially available fluid handlingdevices is not required. As in the example in FIG. 3, a chip may have atleast 246 nanowells, more preferably at least 367 nanowells, and morepreferably at least 45,141 nanowells, and even more preferably, at least90,282 nanowells. While the number of nanowells of the chip may be asmany as 90,282 or more, it is envisioned that the number of nanowellscan usually does not exceed about 1,444,512 nanowells. The number ofnanowells on the preferred embodiment of the chip is sufficient tocontain 82 genes in triplicate along each column and 30,094 genes intriplicate in all rows. As in one such preferred embodiment the numberof wells are sufficient to screen the entire human genome in triplicate.It is envisioned that the number of nanowells on the chip can includeadequate reaction wells for amplifying the entire set of expressed genesin other organisms' genomes as yell.

FIG. 4 is a cross-sectional side view of second layer of an exemplarychip 300 as shown in FIG. 3. FIG. 4 shows a non-limiting example of achip 300 with a thickness of 0.625 mm. FIG. 4 also shows the individualwell 302 dimensions as being 0.25 mm (250 μm) in length and width. Asillustrated, the nanowell depth can be 0.525 mm (525 μm), leaving 0.1 mmof the chip beneath a given well. It is envisioned that nanowellopenings can include a shape such as round, square, rectangle or anyother desired geometric shape. By way of example, a nanowell can includea diameter or width of between about 100 μm and about 1 mm, a pitch orlength of between about 150 μm and about 1 mm and a depth of betweenabout 10 μm to about 1 mm. The cavity of each nanowell make take avariety of configurations. For instance, the cavity within a nanowellmay be divided by linear or curved walls to form separate but adjacentcompartments.

The nanowells of the chip may be formed using commonly knownphotolithography techniques. The nanowells may be formed using a wet KOHetching technique or an anisotropic dry etching technique.

A nanowell of high inner surface to volume ration may be coated withmaterials to reduce the possibility that the reactants contained thereinmay interact with the inner surfaces of the nanowells. A chip can alsobe made of resistive heating material. Non-limiting examples ofmaterials include metal plates such as aluminum and stainless steelsubstrates such as SS-316. Where the substrate used is a metal, it isusually preferable to coat the surface with an insulating layer toprevent corrosion and/or electrophoresis of the sample components duringoperation with fluid samples. Coating is usually not necessary in thecase or non-metal heating material. A variety of protective coatings areavailable, including those made of, for example, SiO2, Si3N4, andTeflon. FIG. 4 shows a chip 300 in which the individual wells 302 areetched with KOH and layered with SiO2.

FIG. 4 also shows an illustrative chip comprising at least two opposingarrays of nanowells. In this figure, the chip 300 has an upper 360 and abottom 362 surface. One of the arrays is arranged along the uppersurface 364 and the other is arranged in an opposite array along thebottom surface 368. The nanowells of the bottom array are positioned inan inverted manner so that the open surface of each unit points awayfrom that of the opposing unit in the chip. The two opposing arrays maybe arranged such that the base of each nanowell is directly opposite tothat of the opposing array.

Though not specifically depicted in FIG. 4, any nanowells in the upper364 and/or bottom 368 arrays may be sealed or unsealed. In addition anynanowell in the upper array may be filled or unfilled, with or withoutthe reaction sample. The subject chip is then placed in thermal contactwith a heating element by placing the chip in contact with an externalheating element.

The surface of a nanowell of a chip of the invention can further bealtered to create adsorption sites for reaction reagents. These sitesmay comprise linker moieties for attachment of biological or chemicalcompound such as a simple or complex organic or inorganic molecule, apeptide, a protein (for example antibody) or a polynucleotide. Oneskilled in the art will appreciate that there are many ways of creatingadsorption sites to immobilize chemical or biological reactants. Forinstance, a wealth of techniques are available for directly immobilizingnucleic acids and amino acids on a chip, anchoring them to a linkermoiety, or tethering them to an immobilized moiety, via either covalentor non-covalent bonds (see, for example, Methods Mol. Biol. Vol. 20(1993), Beier et al., Nucleic Acids Res. 27:1970-1-977 (1999), Joos etal., Anal. Chem. 247:96-101 (1997), Guschin et al., Anal. Biochem.250:203-211 (1997)). The surface of the nanowell can be plasma etched toallow for immobilization of a probe or primer.

As used herein, the term “chemical reaction” refers to any processinvolving a change in chemical properties of a substance. Such process,includes a vast diversity of reactions involving biological moleculessuch as proteins, glycoproteins, nucleic acids, lipids, and inorganicchemicals, or any combinations thereof. The subject chips have a widevariety of uses in chemical and biological applications where differenttemperatures are desired. The chemical reaction may also involveinteractions between nucleic acid molecules, between proteins, betweennucleic acid and protein, between protein and small molecules. Where theprocess is catalyzed by an enzyme, it is also referred to as “enzymaticreaction.”

The subject chips and other apparatus are particularly useful inconducting enzymatic reactions because most enzymes function under onlycertain temperatures. Representative enzymatic reactions that areparticularly temperature dependent include but are not limited tonucleic acid amplification, such as quantitative polymerase chainreaction (qPCR), nucleic acid sequencing, reverse transcription, andnucleic acid ligation. In an embodiment, a nucleic acid amplificationreaction run on a chip of the invention is a real-time polymerase chainreaction. In another embodiment, the nucleic acid amplification reactionis a reverse-transcription coupled polymerase chain reaction.

The chips of the present invention provide a cost-effective means foramplifying nucleic acids. Unlike with conventional microtiter plates andthermal cyclers, the subject chips are highly miniaturized, capable ofperforming rapid amplification of a vast number of target nucleic acidsin small volume, and under independent thermal protocols.

As used herein, “nucleic acid amplification” refers to an enzymaticreaction in which the target nucleic acid is increased in copy number.Such increase may occur in a linear or in an exponential manner.Amplification may be carried out by natural or recombinant DNApolymerases such as Taq polymerase, Pfu polymerase, T7 DNA polymerase,Klenow fragment of E. coli DNA polymerase, and/or RNA polymerases suchas reverse transcriptase.

In general, the purpose of a polymerase chain reaction (PCR) is tomanufacture a large volume of DNA which is identical to an initiallysupplied small volume of target or seed DNA. The reaction involvescopying the strands of the DNA and then using the copies to generateother copies in subsequent cycles. Each cycle will double the amount ofDNA present thereby resulting in a geometric progression in the volumeof copies of the target DNA strands present in the reaction mixture.

General procedures for PCR are taught in U.S. Pat. Nos. 4,683,195(Mullis) and 4,683,202 (Mullis et al.). Briefly, amplification ofnucleic acids by PCR involves repeated cycles of heat-denaturing theDNA, annealing two primers to sequences that flank the target nucleicacid segment to be amplified, and extending the annealed primers with apolymerase. The primers hybridize to opposite strands of the targetnucleic acid and are oriented so that the synthesis by the polymeraseproceeds across the segment between the primers, effectively doublingthe amount of the target segment. Moreover, because the extensionproducts are also complementary to and capable of binding primers, eachsuccessive cycle essentially doubles the amount of target nucleic acidssynthesized in the previous cycle. This results in exponentialaccumulation of the specific target nucleic acids at approximately arate of 2 n, where n is the number of cycles.

A typical conventional PCR thermal cycling protocol comprises 30 cyclesof (a) denaturation at a range of 90° C. to 95° C., (b) annealing at atemperature ranging from 50° C. to 68° C., and (c) extension at 68° C.to 75° C. With the subject chips, the thermal cycling time can bedrastically reduced because of, partly, the small reaction volume, thesmall heating mass, and the design of effective heat dissipationfeatures.

The subject chips can be employed in reverse transcription PCR reaction(RT-PCR). RT-PCR is another variation of the conventional PCR, in whicha reverse transcriptase first coverts RNA molecules to double strandedcDNA molecules, which are then employed as the template for subsequentamplification in the polymerase chain reaction. In carrying out RT-PCR,the reverse transcriptase is generally added to the reaction sampleafter the target nucleic acids are heat denatured. The reaction is thenmaintained at a suitable temperature (for example, 30-45° C.) for asufficient amount of time (for example, 5-60 minutes) to generate thecDNA template before the scheduled cycles of amplification take place.Such reaction is particularly useful for detecting the biological entitywhose genetic information is stored in RNA molecules. Non-limitingexamples of this category of biological entities include RNA virusessuch as HIV and hepatitis-causing viruses. Another important applicationof RT-PCR embodied by the present invention is the simultaneousquantification of biological entities based on the mRNA level detectedin the test sample. One of skill in the art will appreciate that if aquantitative result is desired, caution must be taken to use a methodthat maintains or controls for the relative copies of the amplifiednucleic acids.

Methods of “quantitative” amplification of nucleic acids are well knownto those of skill in the art. For example, quantitative PCR (qPCR) caninvolve simultaneously co-amplifying a known quantity of a controlsequence using the same primers. This provides an internal standard thatmay be used to calibrate the PCR reaction. Other ways of performing qPCRare available in the art.

Nucleic acid amplification is generally performed with the use ofamplification reagents. Amplification reagents typically includeenzymes, aqueous buffers, salts, primers, target nucleic acid, andnucleoside triphosphates. Depending upon the context, amplificationreagents can be either a complete or incomplete amplification reactionmixture.

Reagents contained within a chip of the invention depend on the reactionthat is to be run. In an embodiment, at least one of the units of thearray of addressable units contains a reagent for conducting the nucleicacid amplification reaction. Reagents can be reagents for immunoassays,nucleic acid detection assays including but not limited to nucleic acidamplification. Reagents can be in a dry state or a liquid state in aunit of the chip.

In an embodiment, at least one of the units of the array of addressableunits of a chip capable of carrying out a nucleic acid amplificationreaction contains at least one of the following: a probe, a polymerase,and a dNTP. In another embodiment, the nanowells of a chip contain asolution comprising a probe, a primer and a polymerase. In variousembodiments, each chamber comprises (1) a primer for a polynucleotidetarget within said standard genome, and (2) a probe associated with saidprimer which emits a concentration dependent signal if the primer bindswith said target.

In various embodiments, each unit comprises a primer for apolynucleotide target within a genome, and a probe associated with theprimer which emits a concentration dependent signal if the primer bindswith the target.

In another embodiment, at least one unit of the chip contains a solutionthat comprises a forward PCR primer, a reverse PCR primer, and at leastone FAM labeled MGB quenched PCR probe.

In an embodiment, primer pairs are dispensed into a unit and then dried,such as by freezing. The user can then selectively dispense, such asnano-dispense, the sample, probe and/or polymerase.

In other embodiments of the invention, the nanowells may contain any ofthe above solutions in a dried form. In this embodiment, this dried formmay be coated to the wells or be directed to the bottom of the well. Theuser can add a mixture of water and the sample to each of the wellsbefore analysis.

In this embodiment, the chip comprising the dried down reaction mixturemay be sealed with a liner, stored or shipped to another location. Theliner is releasable in one piece without damaging the adhesiveuniformity. The liner is visibly different than the cover to aid inidentification and for ease of handling. The material of the liner ischosen to minimize static charge generation upon release from theadhesive. When the user is ready to use the chip, the seal is broken andthe liner is removed and the sample is added to the units of the chip.The chip can then sealed and placed into contact with a heating element.

In many applications, sealing the units (for example, nanowells) isdesirable to prevent evaporation of liquids and thus maintains thepreferred reaction concentrations throughout the thermal cycling.

The chip may be used for genotyping, gene expression, or other DNAassays preformed by PCR. Assays performed in the plate are not limitedto DNA assays such as Taqman, Invader, Taqman Gold, SYBR gold, and SYBRgreen but also include other assays such as receptor binding, enzyme,and other high throughput screening assays. In some embodiments, a ROXlabeled probe is used as an internal standard.

The invention also provides a method for performing a PCR analysis usinga chip comprising a plurality of preloaded nanowells, the methodcomprising: placing a sample into the nanowells to create a reactionmixture; sealing the nanowells of the chip with mineral oil or anothersealing mechanism; placing the chip into a thermal cycling system;cycling the system; and analyzing results:

In accordance with the present invention, the units of the chip comprisea solution operable to perform multiplex PCR. In a preferableembodiment, the units are capable of having multiple PCR reactions ineach individual unit based on the chemistry and the probes that areincluded in the solution. “Multiplex PCR” is the use of more than oneprimer pair in the same unit. This method can be used for relativequantitation where one primer pair amplifies the target and anotherprimer pair amplifies the endogenous reference. A multiplex reaction canbe performed using a variety of methods including the Standard CurveMethod or the Comparative Ct Method.

Various probes can be used, such as FAM which is a carboxy-fluoresceinwhich has an excitation wavelength from about 485 nm and an emissionwavelength from about 510-520 nm; SYBR Green 1 which is normally usedfor RT-PCR and has an excitation wavelength of about 488 nanometers andan emission wavelength of about 510 nanometers; TET which has anemission wavelength from about 517 nanometers to about 538 nanometers;the probes from the group of HEX, JOE and VIC, which have emissionwavelengths from 525-535 mm to about 546-556 nm; TAMRA which is acarboxy-tetra methylrhodamine, and has an emission wavelength from about556 nanometers to about 580 nanometers; ROX which is acarboxy-x-rhodamine, which has an emission wavelength from about 575-585nm to about 605-610 nm; ALEXA, which has an emission range from about350 nanometers to about 440 nanometers; TEXAS RED, which has an emissionwavelength from about 580-585 nm to about 600-610 nm; Cy3, which has anemission wavelength of about 545 nanometers to about 568 nanometers;Cy5, which has an emission wavelength of about 635-655 nm to about665-675 nm; Cy7, which has an emission wavelength of about 715nanometers to about 787 nanometers. Optimized interference filtersprecisely match the excitation and emission wavelengths for eachfluorophore to block out unwanted cross-talk from spectrally adjacentfluorophores.

The choice of primers for use in nucleic acid amplification will dependon the target nucleic acid sequence. Primers used in the presentinvention are generally oligonucleotides, usually deoxyribonucleotidesseveral nucleotides in length, that can be extended in atemplate-specific manner by the polymerase chain reaction. The design ofsuitable primers for amplifying a target nucleic acid can be determinedby one skilled in the art.

For a convenient detection of the amplified nucleic acids resulting fromPCR or any other nucleic acid amplification reactions described above orknown in the art, primers may be conjugated to a detectable label.Detectable labels suitable for use in the present invention include anycomposition detectable by spectroscopic, photochemical, biochemical,immunochemical, electrical, optical or chemical means. A wide variety ofappropriate detectable labels are known in the art, which includeluminescent labels, enzymatic or other ligands. In preferredembodiments, one will likely desire to employ a fluorescent label or anenzyme tag, such as digoxigenin, β-galactosidase, urease, alkalinephosphatase or peroxidase, avidin/biotin complex.

The labels may be incorporated by any of a number of means well known tothose of skill in the art. In one aspect, the label is simultaneouslyincorporated during the amplification step. Thus, for example, PCR withlabeled primers or labeled nucleotides can provide a labeledamplification product. In a separate aspect, transcription reaction inwhich RNA is converted into DNA, using a labeled nucleotide (for examplefluorescein-labeled UTP and/or CTP) or a labeled primer, incorporates adetectable label into the transcribed nucleic acids.

The invention also provides reagents and kits suitable for carrying outpolynucleotide amplification methods of this invention. Such reagentsand kits may be modeled after reagents and kits suitable for carryingout conventional PCR, RT-PCR, and other amplification reactions. Suchkits comprise a chip of the invention and a reagent selected from thegroup consisting of an amplification reagent, a detection reagent andcombinations thereof. The kits may comprise reagents packaged fordownstream or subsequent analysis of the multiplex amplificationproduct. The primers included in the individual units can, independentlyof one another, be the same or a different set of primers comprising theplurality of multiplex amplification primers.

In another embodiment, the oligonucleotide probes are suitable fordetecting single nucleotide polymorphisms, as is well-known in the art.A specific example of such probes includes a set of four oligonucleotideprobes which are identical in sequence save for one nucleotide position.

Each of the four probes includes a different nucleotide (A, G, C andT/U) at this position. The probes may be labeled with labels capable ofproducing different detectable signals that are distinguishable from oneanother, such as different fluorophores capable of emitting light atdifferent, spectrally resolvable wavelengths (for example, 4 differentlycolored fluorophores).

The primer pairs used in this invention can be obtained by chemicalsynthesis, recombinant cloning, or a combination thereof. Methods ofchemical polynucleotide synthesis are well known in the art and need notbe described in detail herein. One of skill in the art can use thetarget sequence to obtain a desired primer pairs by employing a DNAsynthesizer or ordering from a commercial service.

Nucleic acid amplification requires a target nucleic acid in a buffercompatible with the enzymes used to amplify the target. The targetnucleic acid used for this invention encompasses any reaction samplessuspected to contain the target sequence. It is not intended to belimited as regards to the source of the reaction sample or the manner inwhich it is made. Generally, the test sample can be biological and/orenvironmental samples. Biological samples may be derived from human,other animals, or plants, body fluid, solid tissue samples, tissuecultures or cells derived therefrom and the progeny thereof, sections orsmears prepared from any of these sources, or any other samplessuspected to contain the target nucleic acids. Preferred biologicalsamples are body fluids including but not limited to blood, urine,spinal fluid, cerebrospinal fluid, sinovial fluid, ammoniac fluid,semen, and saliva. Other types of biological sample may include foodproducts and ingredients such as vegetables, dairy items, meat, meatby-products, and waste. Environmental samples are derived fromenvironmental material including but not limited to soil, water, sewage,cosmetic, agricultural and industrial samples.

Preparation of Nucleic Acids Contained in the Test Sample can be CarriedOut According to Standard Methods in the art or procedures described.Briefly, DNA and RNA can be isolated using various lytic enzymes orchemical solutions according to the procedures set forth in Sambrook etal. (“Molecular Cloning: A Laboratory Manual”), or extracted by nucleicacid binding resins following the accompanying instructions provided bymanufacturers' instructions.

The nucleic acid in the reaction sample can be cDNA, genomic DNA orviral DNA. However, the present invention can also be practiced withother nucleic acids, such as mRNA, ribosomal RNA, viral RNA. Thesenucleic acids may exist in a variety of topologies. For example, thenucleic acids may be single stranded, double-stranded, circular, linearor in form of concatamers. Those of skill in the art will recognize thatwhatever the nature of the nucleic acid, it can be amplified merely bymaking appropriate and well recognized modifications to the method beingused.

In an aspect of the invention, a chip for running a reaction comprisesan array of addressable units each being configured to run a chemicalreaction. The addressable units of the chip are arranged according to apredetermined set of temperatures for running the chemical reactions ofthe units. At least one of the units is addressed to indicate thepredetermined temperature for running the chemical reactions within theunit.

In an embodiment, a plurality of the units are addressed to indicate thepredetermined temperature for running the chemical reactions. In anotherembodiment, each of the units is individually addressed to indicate thepredetermined temperature for running the chemical reaction within saidunit.

The predetermined temperature of a unit of a chip of the invention canbe configured to yield at least 90% of homogeneous product from thechemical reaction. If the annealing temperatures are optimized, theconfiguration of an addressable chip based upon predetermined reactiontemperatures can significantly improve the yield and quality of areaction product. This can be important when a user is interested inanalyzing a large number of nucleic acids, such as the whole genome ofan organism, with a chip and the apparatuses of the present invention.In an embodiment, the improved reaction yield plays an important role inthe use of a chip, apparatus, or system of the invention when used as amedical diagnostic instrument.

FIG. 5 illustrates an example chip 500 of the invention comprising a setof smaller chips 510. The set of smaller chips 510, also referred to asa plate 500, are placed in contact with a thermally insulative frame530. Each smaller chip 510 represents a different temperature zone, aslabeled in FIG. 5, in this example, zones 1, 2, 3, 4, 5, and 6. In anembodiment, the temperature zones correspond to a predetermined set ofannealing temperatures for conducting polymerase chain reaction withinthe nanowells 520 of the chip 500. By separating the overall chip 500into different temperature zones, a user can increase the specificity ofthe reaction within the nanowells 520. For example, when analyzing anentire genome of a species, a plurality of temperature zones can allowfor more accurate determination of the genome.

The subject chips can contain one or more grooves etched in at thebottom side of the chip. In general, the grooves are under-trenches,open channels or paths to allow air passage. The grooves reduce thethermal mass of the chip, increase the surface area, and thus enhancethe thermal performance of the chips. The grooves can be fabricated inany shapes, including but not limited to circular, elliptical, oval,conical, rectangular, triangular, and polyhedral. The grooves may befurther divided by linear or curved walls to form separate but adjacentchannels, or by circular walls to form inner and outer annular channels.The dimensions of the grooves will depend on the overall sizes anddepths of the chips. The depths of the grooves may range from about onetenth to about nine tenths of the chip depths. The other dimensions,namely widths and lengths, may be shorter, longer or comparable to thecorresponding dimensions of the chips. In particular, the L-shapedgrooves surround the base of a unit. As the air flows through thepassageways formed by any of the grooves, it removes heat from thesurfaces of unit by passive heat dissipation, thus increasing the speedof thermal cycling.

Several factors apply to the selection of a suitable chip substrate. Thesubstrate is often a good thermal conductor. A good thermal conductorgenerally has a thermal conductivity value higher than 1 W/m⁻¹K⁻¹,preferably higher than 100 W/m⁻¹ K⁻¹, more preferably higher than 140W/m⁻¹K⁻¹. Whereas the material's thermal conductivity may be 250W/m⁻¹K⁻¹ or higher, it usually does not exceed 500 W/m⁻¹K⁻¹. Second, thesubstrate must be relatively inert and chemically stable. Such substrategenerally exhibits a low level of propensity to react with the reactionsamples employed in the intended application. Moreover, the materialsshould also be selected based upon the ability or feasibility tointegrate the thermal control elements onto or adjacent to them. Avariety of materials meet these criteria. Exemplary materials includebut are not limited to metalloids or semiconductors, such as silicon,silicates, silicon nitride, silicon dioxide, gallium phosphide, galliumarsenide, or any combinations thereof. Other possible materials areglass, ceramics (including crystalline and non-crystalline silicate, andnon-silicate-based ceramics), metals or alloys, composite polymers thatcontain dopants (for example, aluminum oxide to increase thermalconductivity), or any of a range of plastics and organic polymericmaterials available in the art. In one embodiment, the nanowells arefabricated in such substrates including Al or SS-316 as well as similarothers.

In an embodiment, the chips are fabricated using a thermally conductivepolymer. For example, the chips can be fabricated using polycarbonate,polypropylene, or any other conductive polymer known to those with skillin the art.

The chips can be fabricated by any method as would be obvious to oneskilled in the art. Examples of method of making a chip of the inventioninclude, but are not limited to, micro drilling, electric dischargemethod, hot embossing, and hot embossing with a tool made from whichuses water as light guide.

Alternatively, chips of the present invention can be fabricated usingtechniques well established in the Integrated Circuit (IC) andMicro-Electro-Mechanical System (MEMS) industries. The fabricationprocess typically proceeds with selecting a chip substrate, followed byusing appropriate IC processing methods and/or MEMS micromachiningtechniques to construct and integrate various components.

Fabrication of the subject chips can be performed according to standardtechniques of IC-processing and/or MEMS micromachining. The subjectchips can be fabricated as multi-layer structures. The process generallyproceeds with constructing the bottom layer. Then a combination oftechniques including but not limited to photolithography, chemical vaporor physical vapor deposition, dry or wet etching are employed to buildstructures located above or embedded therein. Vapor deposition, forexample, enables fabrication of an extremely thin and uniform coatingonto other materials, whereas etching allows for mass production oflarger chip structures. Other useful techniques such as ionimplantation, plasma ashing, bonding, and electroplating can also beemployed to improve the surface properties of the chips or to integratevarious components of the chips. The following details the fabricationprocess with reference to the exemplary chip designs depicted in thefigures. The same general process and the apparent variations thereofare applicable to fabricate any of the subject chips described herein.

FIG. 4 is a cross-section view of a portion of an exemplary chip design.In this embodiment, the nanowell is embedded within a body which is madeup of first and second (or bottom and top) layers of substratesrespectively. The process begins with providing a first layer ofsubstrate which is generally a heat resistant material such as glass,Pyrex wafer, or any other suitable materials described herein or knownin the art. The next step is to create the nanowell that forms the basisof the unit. The nanowell is generally disposed within the second layerthat is typically a silicon wafer (see, for example, FIG. 4). Thesilicon wafer may go through several processing steps prior to beingattached to the first layer. For example, the silicon wafer may beattached to a layer of photoresist to render the surface moresusceptible to chemical etching after exposure to UV light during theprocess of photolithography. The layer of photoresist defines, byprecise alignment of the photo-mask, the size and location of thenanowell that is to be formed by a subsequent etching step. The siliconwafer is then etched by a variety of means known in the art to form thewell cavity. A commonly practiced etching technique involves the use ofchemicals, for example, potassium hydroxide (KOH), which removes thesilicon wafer to form the desired shape.

Once the nanowells of the subject chips are fabricated, their surfaceproperties can be improved to suit the particular application. Wherelarge surface area is desired, the wall of the nanowell may be furtheretched by, for example, a plasma etcher to obtain very fine dendrites ofsilicon, commonly referred to as “black silicon”. The presence of blacksilicon can dramatically increase the effective heating surface area.The black silicon fabricated at the base of the nanowell may also serveas an anchor for photon-sensing devices, temperature sensors and othercontrol elements.

As discussed herein, a nanowell of high inner surface to volume ratiomay be coated with materials to reduce the possibility that thereactants contained therein may interact with the inner surfaces of thewell. The choice of methods for applying the coating materials willdepend on the type of coating materials that is used. In general,coating is carried out by directly applying the materials to thenanowell followed by washing the excessive unbound coating material.Certain coating materials can be cross-linked to the surface viaextensive heating, radiation, and by chemical reactions. Those skilledin the art will know of other suitable means for coating a nanowellfabricated on chip, or will be able to ascertain such, without undueexperimentation.

Sample preparation then includes combining the PCR reaction samplemixture, the labeled primers, and the sample with a drop of oil whichcan be individually nano dispensed to prevent evaporation. The sample isthen dispensed into the individual nanowells using a piezo dispenser anda dew point dispensing technique.

To prevent evaporation of aqueous reaction samples, the samples can beapplied to the nanowell at or around dew point. As used herein, “dewpoint” refers to a temperature range where the droplet size does notchange significantly. At dew point, an equilibrium is reached betweenthe rate of evaporation of water from the sample droplet and the rate ofcondensation of water onto the droplet from the moist air overlying thechip. When this equilibrium is realized, the air is said to be saturatedwith respect to the planar surface of the chip. At one atmosphericpressure, the dew point is about 14° C. Accordingly, dispensing aqueousreaction samples is preferably carried out at a temperature no more thanabout 1° C. to about 5° C. degrees above dew point. As is apparent toone skilled in the art, dew point temperature increases as the externalpressure increases. Therefore, where desired, one may dispense thereaction samples in a pressured environment to prevent evaporation.

Amplified nucleic acids present in the subject chips may be detected bya range of methods including but not limited to (a) forming a detectablecomplex by, for example, binding the amplified product with a detectablelabel; and (b) electrophoretically resolving the amplified product fromreactants and other components of the amplification reaction.

In certain embodiments, the amplified products are directly visualizedwith detectable label such as a fluorescent DNA-binding dye. Because theamount of the dye intercalated into the double-stranded DNA molecules istypically proportional to the amount of the amplified DNA products, onecan conveniently determine the amount of the amplified products byquantifying the fluorescence of the intercalated dye using the opticalsystems of the present invention or other suitable instrument in theart. DNA-binding dye suitable for this application include SYBR green,SYBR blue, DAPI, propidium iodine, Hoeste, SYBR gold, ethidium bromide,acridines, proflavine, acridine orange, acriflavine, fluorcoumanin,ellipticine, daunomycin, chloroquine, distamycin D, chromomycin,homidium, mithramycin, ruthenium polypyridyls, anthramycin, and thelike.

In addition to various kinds of fluorescent DNA-binding dye, otherluminescent labels such as sequence specific probes can be employed inthe amplification reaction to facilitate the detection andquantification of the amplified product. Probe based quantitativeamplification relies on the sequence-specific detection of a desiredamplified product. Unlike the dye-based quantitative methods, itutilizes a luminescent, target-specific probe (for example, TaqMan®probes) resulting in increased specificity and sensitivity. Methods forperforming probe-based quantitative amplification are well establishedin the art and are taught in U.S. Pat. No. 5,210,015.

In various embodiments, the chip additionally comprises alignmentfeatures, operable to align or attach a cover to the chip or to align orattach the chip to a heating element. In various embodiments, suchfeatures comprise concave or convex features. Such features includepins, ridges, snaps, screws, and combinations thereof.

In various embodiments, the chip assembly comprises a temperaturecontrol element, which facilitates the monitoring or control of thetemperature of reaction chambers. Such temperature control elementsinclude but are not limited to channels or other structures thatfacilitate the flow of a heating or cooling gas through the assembly.

In another aspect of the invention, a method of conducting a reactionthat involves a plurality of reaction samples and requires cycling atleast two temperature levels is disclosed and comprises: providing achip comprising an array of addressable units each being configured torun a chemical reaction, wherein the units are arranged according to apredetermined set of temperatures for running the chemical reactions ofthe units, such that at least one of the units is addressed to indicatethe predetermined temperature for running the chemical reactions withinsaid unit; placing the plurality of reaction samples into the units ofthe chip according to the set of predetermined temperatures; andcontrolling a heating element to effect cycling at least two temperaturelevels. In one aspect a method of conducting a chemical reaction caninclude providing a chip as described herein where the chip isconfigured to receive a reaction sample. A heating element can beprovided that is positionable in relation to the chip to provide thermalcontact between at least one heating element and the chip. A chemicalreaction can be conducted in the reaction sample by varying thetemperature of the chip. Varying the temperature in the chip can beeffected, for example, by moving at least one heating element inrelation to the chip such that the heating element is in thermal contactwith the chip.

The method uses a chip of the invention and a thermal cycling apparatusor the invention. Typically, in the analysis of an entire genome of anorganism, amplification of different genes or nucleic acid sequences areoptimized at different annealing temperatures. These different annealingtemperatures could be grouped into general zones to improve thesensitivity and specificity of an assay. A chip of the invention cantherefore be addressed according to one of the annealing temperaturezones.

The selection of the annealing temperature can be a critical componentfor optimizing the specificity of a PCR reaction. The annealingtemperatures of different sequences for nucleic acid amplification canbe determined in a variety of ways. One method is to determine themelting temperature (T_(m)) of a nucleic acid sequence. The meltingtemperature is the temperature at which one half of the DNA duplex willdissociate and become single stranded. When designing a nucleic acidamplification, primer length and sequence are important in designing theparameters of a successful amplification. For example, the meltingtemperature of a DNA increases both with its length, and with increasingguanine and cytosine content which can be approximated using a simpleformula.

The annealing temperature chosen for a PCR reaction depends directly onlength and composition of the primer(s). The annealing temperature(T_(a)) can be chosen to be about 5° C. below the lowest T_(m) of thepair of primers to be used (Innis and Gelfand, 1990). Another examplemethod of calculating the annealing temperatures is:

T _(a)Opt=0.3×(T _(m) of primer)+0.7×(T _(m) of product)−25

where T_(m) of primer is the melting temperature of the less stableprimer-template pair and T_(m) of product is the melting temperature ofthe PCR product (Rychlik, et al., 1990).

If the annealing temperature of a PCR reaction is too low, a primer cananneal to sequences other than the true target, and can lead tonon-specific amplification and consequent reduction in yield of thedesired product. A consequence of too high an annealing temperature isthat too little product will be made, as the likelihood of primerannealing is reduced. Another consideration is that a pair of primerswith very different annealing temperatures may never give appreciableyields of a unique product, and may also result in inadvertentasymmetric or single-strand amplification of the most efficiently primedproduct strand.

In PCR thermal cycling, the optimum T_(a) and the maximum, T_(a) rangefor each different primer pair and can vary from gene to gene in acontinuous fashion. Typically, the anneal temperature to produce highquality anneal for a given primer pair is in a range of 1 to 3° C. Ifthe anneal temperature is higher, the PCR anneal step can slow down; ifit is lower, the primers might bind to the DNA at sites other than thedesired ones or to other species.

One method is to “bin” the gene assays; the temperature of a given assayis assigned to the addressable portion of the chip with the temperatureclosest to that of T_(a) of that given gene assay. For example, if theapparatus or chip has 3 temperature zones of 60, 62.5 and 65° C., anassay that has an optimum behavior T_(a)=64° C. is assigned to the 65°C. zone.

The annealing step of a PCR reaction typically occurs in 40 sec or less,depending partially on the length of the sequence to be amplified.

While the PCR is normally started at 5° C. below the calculatedtemperature of the primer melting point (T_(m)), the optimum temperatureoften can be much higher than the calculated temperature. In someembodiments, the annealing temperature must be empirically tested. Forexample, multiple PCR reactions with gradually increasing temperaturescan be carried out until the optimal annealing temperature isdetermined.

Many of the genetic sequences in a genome can be grouped intotemperature zones. In an embodiment, the units of a chip of theinvention represent different annealing temperature zones, for example,the zones are 2° C. increments over the range of 54 to 68° C. The unitsare then addressed according to predetermined temperature at which theannealing step of the PCR reaction will occur.

After a unit is addressed according to a predetermined temperature,sample can then be dispersed into the chip. The sample can be added tothe chip by a variety of methods as disclosed herein.

In various embodiments, a system or chip can additionally comprise afilling device, which is operable to facilitate filling of amplificationreagents or samples into the addressable units of the chip. Fillingdevices among those useful herein include physical and chemicalmodalities that direct, channel, route or otherwise effect fillingreagents or samples into the addressable units.

In various embodiments, the chip may comprise raised or depressedregions, for example, features such as barriers and trenches to aid inthe distribution and flow of liquids on the surface of the chip. In anembodiment, the filling system comprises capillary channels. Thedimensions of these features are flexible, depending on factors, such asavoidance of air bubbles upon assembly and mechanical convenience andfeasibility.

The filling system comprises any apparatus which facilitates theplacement of amplification reagents or sample on the surface of thechip, preferably effecting placement of such reagents or sample inaddressable units. Such apparatus among those useful herein includedevices for pouring of reagents or samples onto the surface so as tosubstantially cover the entire surface. In an embodiment the fillingsystem comprises a device for pipetting, spotting or spraying ofreactants to specific reaction chambers (for example, by usepiezoelectric pumps). The filling system can be a nano-dispenser. Inanother embodiment, the filling apparatus comprises a vacuum pumpoperable to fill the reaction chambers of the chip. Filling systems mayalso include devices for applying centrifugal force to the chip. In oneembodiment, the filling system is in close proximity to or in fluidcommunication with a filling device in the chip. The filling system canoperate automatically or according a protocol from a computer.

APPARATUS

One aspect of the present invention is the design of an apparatusconfigured to receive a miniaturized chip designed for the multiplexedanalyses of individual molecules, and/or simultaneous performance of avast number of chemical reactions. In one embodiment, the presentinvention provides a highly automated, miniaturized, analyticalinstrument that allows manipulations with precise control oftemperature, evaporation, small-volume reagent delivery, and/or productdetection in a multiplexed fashion.

The apparatus of one aspect of the invention includes at least oneheating element useful for heating or cooling a chip. It is to beunderstood that where the heating element is configured to reduce thetemperature of a chip, the component functions essentially as a coolingelement.

In one embodiment the apparatus includes a base with at least oneheating element positioned on the base. The heating element can providea range of useful temperatures. For example, the heating element can beconfigured to provide a temperature in the range between about −20° C.to about 120° C. as desired.

The heating element can be configured to receive a chip. In variousembodiments the heating element can be positioned either below the chipor above the chip. In a particular embodiment where only one heatingelement is present, the heating element can move relative to the chip.Where desired, the chip can be stationary.

FIG. 6 is a non-limiting example of a schematic diagram of the oneembodiment of the apparatus. FIG. 6 depicts a base 600 with at least oneheating element 602 positioned on the base 600. The heating element 602is adapted to receiving a chip 650, such chip is capable of containingand confining a reaction sample in one of the nanowells 652 on the chip650. The heating element 602 of the apparatus can be moved in twopositions relative to the chip 650.

The heating element 602 as seen in FIG. 6 can be divided into differenttemperature zones as seen in FIG. 7. FIG. 7 is an illustrative drawingof a heating element positioned below the chip 700 used to controland/or vary the temperature of a chip. In one embodiment the temperatureranges for a heating element can be in the range of 90° C. to 95° C. forthe denaturation temperature, 52° C. to 65° C. for the primer annealingstage, and 68° C. to 75° C. for the primer-dependent extension stage. Anon-limiting example of a heating element 700 is shown in FIG. 7. Theheating element 700 in this example is one with temperature zones of 95°C. (702), 52° C. (704), and 72° C. (706) for the denaturation, primerannealing stage and primer-dependent extension stage of PCR,respectively. The dimensions of each temperature zone of the heatingelement 700 can vary in size. In one embodiment depicted in FIG. 7, theheating element positioned below an example of the chip is about 9inches in length by 9 inches in width.

A chip adapted to containing and confining a reaction sample is thenplaced on the heating element 700 of the apparatus adapted to receivingthe chip. Using the heating element positioned below 700 the chip ofFIG. 7, the heating elements then physically moved so that the chipcomes in contact with the 95° C.-block, followed by the 52° C.-block,and then the 72° C.-block. The movement of the heating elementrespective to the chip allows the critical ramp down rates to be a lotfaster due to the larger surface area on the heating element. The largersurface area also allows for cooling from the backside of the chip dueto transfer of the chip from temperature to temperature. This techniquealso allows the chip to be heated and cooled at a much faster rate thanthe traditional way of simply cooling the chip between cycles.

Another method for controlling and/or varying the temperature of thechip 800 is depicted in FIG. 8. FIG. 8 is a non-limiting example of theheating element 850 positioned above the chip 800. As shown in FIG. 8,the heating element can be a series of on/off arc halogen lamps 852positioned above the chip 800. Heat reflectors 854 are positioned aroundeach halogen lamp 852 to ensure a uniform heat source from the heatlamps 852. The high output power of the arc lamp 852 and the spectralmatch of the output to silicon provides a very fast “ramp up” rate ofthe arc lamps 852. The fact that the lamp is an arc lamp means thatswitch off of the arc lamp 852 is virtually instantaneous. FIG. 8 alsoshows that the chip 800 can also be sealed with a radiation curableadhesive 802 to help prevent evaporation of the reaction sample.Following a reaction cycle, in one non-limiting example the chip is thenscanned with a scanner 860, for example, a hyper spectral or CCDScanner. In certain aspects, such an optically coupled system transmitsexcitation beams into the wells containing the reaction samples at aplurality of times during the amplification, and monitors the opticalsignals coming from the nanowells at each of the plurality of timesbetween cycles.

FIG. 9 graphically shows the change in temperature in Celsius (y-axis)versus time in seconds (x-axis) of the on/off arc lamps. As seen in FIG.9, the halogen lamps ramp up rate of the arc lamp is 20° C. to 100°C./sec for a temperature of 95+/−0.5° C. in less than about 2 secondsand has a 20° C. to 100° C. ramp down rate to 65+/−0.5° C., also in lessthan about 2 seconds. The temperature of the arc lamp shifts fromapproximately 65° C. to approximately 95° C. during the PCR reactionprocess.

Where desired, the subject devices are designed to minimize the movementof pulsed heat into the nanowells fabricated out of Aluminum/Silicon inan alternative chip. One way to minimize the amount of pulsed heat is toreduce the time that the Al/Si spends at the highest temperatures andthereby reducing “thermal budget”. Speeding the “ramp-up” and“cool-down” rates and providing the fastest possible transition fromheating to cooling, or “turn around” is therefore important. This methodof heating and cooling the chip also ensures uniformity in the heatingof the chip. Such uniformity prevents stresses due to temperaturedifferences of a few degrees that can lead to variations in theperformance of the nanowells.

As seen in FIG. 10, there are several temperature output profiles thatthe arc lamps are capable of delivering. The graphs along row (a) showthe change in temperature (y-axis) versus the change in time (x-axis).Correspondingly, the graphs along row (b) depict the chip response tothe rapid thermal processing (RTP) profiles depicted in row (a). Inresponse to a slower change in temperature over time, such as in a spikeor impulse optical heater profile, the chip temperature remains constantover the entire depth of the chip. In response to a more rapid change intemperature over time, such as in a laser or flash assist heaterprofile, the temperature of the chip varies depending on the depth ofthe chip relative to the heating source.

Without being limited to a particular theory, FIG. 10 illustrates anumber of temperature output profiles corresponding to the invention.The spike heater profile, as seen in FIG. 10( a)(1), which ischaracterized by a rounded thermal profile, generates a chip responsetemperature similar to the heating source temperature, as seen in FIG.10( b)(1). In the impulse heater profile (FIG. 10( a)(2)), characterizedby a peaked thermal profile, the temperature of the heat source isfaster than the temperature of the chip n response to the heat source,but the temperature of the chip is still kept relatively uniform overthe entire depth of the chip relative to the distance from the heatsource (FIG. 10( b)(2)). For a laser heater profile as seen in FIG. 10(a)(3), characterized by a sharply peaked profile, the heat source actsmuch faster than the chip. Therefore, only the surface layer of the chipis heated (FIG. 10( b)(3)). The deeper the depth of the chip, the coolerthe temperature profile. Finally, for the flash assist profile (FIG. 10(a)(4)), characterized by initial bulk heating followed by a flash forsurface annealing, the chip response is similar to the chip response tothe laser profile (FIG. 10( b)(4)).

In another aspect of the invention, an apparatus is described forconducting a chemical reaction requiring cycling at least twotemperature levels, that comprises: a chip for running a reactioncomprising an array of addressable units each being configured to run achemical reaction, wherein the units are arranged according to apredetermined set of temperatures for running the chemical reactions ofthe units, such that at least one of the units is addressed to indicatethe predetermined temperature for running the chemical reactions withinsaid unit; and a heating element in thermal contact with the chip.

In a preferable embodiment, the addressable units of the chip areconfigured to run a nucleic acid amplification reaction, including butnot limited to real-time PCR. The units can be nanowells that comprise areagent, a probe, a primer, a dNTP, or a combination thereof. Thepredetermined temperatures can be different annealing temperatures forcarrying out a PCR reaction.

The heating element can be a simple heater, such as a plate comprising aresistive heater or a thermoelectric heater, or an elaborate thermalcycling apparatus. In some examples, the heating element is thermalcontact with a heat sink to allow for rapid temperature changes of thechip when the chip is in thermal contact with the heating element. A fancould also be coupled to the heating element to provide more controlover rapid thermal cycling. Other examples of heating elements includethin film heaters that can be heated rapidly by conduction or haveelectromagnetic heaters incorporated into the film. A materialparticularly suitable for fabricating the thin film heaters is indiumtin oxide (ITO). ITO is a transparent ceramic material with a very highelectrical conductivity. Because ITO can be prepared in bulk or in formof thin layer, it is particularly useful as either an integral or anexternal heating element.

In another embodiment, heating elements are compatible to the chips interms of size and configuration. In an embodiment, the apparatus furthercomprises a plurality of heating elements. The heating element can beplaced as a detachable unit adjacent to, at the base and/or on top ofthe chip. In a preferable embodiment, the heating element area issignificantly larger than the chip area, in order to minimize edgeeffects at the edges of a heating element.

Some examples of heating element construction and setup are illustratedin FIGS. 11 a-c. In these examples, the heating elements 1100 arecoupled to a heat sink 1110 and a fan 1120 to improve control overtemperature changing and ramp up times for thermal cycling. A metalthermal block 1130 can be used between the heating element 1100 and thesample (or chip) 1140 that is in intimate thermal contact with theelement 1100 and the chip 1140. The block 1130 can have a high thermalconductivity even if the chip 1140 might not have a high thermalconductivity, to produce a reproducible temperature change. If multipleheating elements are used to define a temperature zone or multipletemperature zones, any small disturbance of the heating (for example, anair breeze) can produce a significant change in temperature. If theresistance is made low using a thermal block, the transfer of heat fromzone to zone can be smoothed. In addition, the metal thermal block canhold temperature sensors on the top surface. The temperature sensor cancorrespond to each zone, or can be part of the chip itself. The thermalblock can also provide vacuum channels to allow vacuum to hold the chipin intimate thermal contact with the block and/or heating elements. Asdemonstrated in FIGS. 11 a-c, the heating element 1100 can comprise oneor a plurality of thermoelectric 1150 or resistive 1160 heatingelements, or a combination of both.

The heating element can provide a temperature gradient. The temperaturegradient herein can be a temperature that is higher in a portion thananother portion across a single heating element. For example, aresistive or thermoelectric heater can be configured to create a thermalgradient across the heating element. The thermal gradient can also bedefined as a temperature that is higher in one portion than anotherportion of the chip. For example, if a plurality of heating elements areused, one heating element could deliver a higher temperature to one sideor end of the chip and a second heating element could deliver adifferent temperature to one side or end of the chip, thereby creating athermal gradient.

The apparatus can further comprise an optical system operatively coupledto the chip, wherein the optical system detects an optical signal comingfrom a unit. In one exemplary embodiment, the chip, heating element, andoptical system make up a system of the invention.

In an aspect, the invention disclosure includes an apparatus forconducting a chemical reaction involving cycling at least twotemperature levels comprising: a body configured to receive a sampleholder for containing the chemical reaction; a first heater comprising aplurality of temperature zones; and a second heater providing a uniformtemperature, wherein the first heater and the second heater areconfigured to be movable between a first and a second orientation, andwherein the first orientation places the heater in thermal contact withthe sample holder and the second orientation does not place the heaterin thermal contact with the sample holder.

The sample holder can be a chip for running a reaction comprising anarray of addressable units each being configured to run a chemicalreaction, wherein the units are arranged according to a predeterminedset of temperatures for running the chemical reactions of the units,such that at least one of the units is addressed to indicate thepredetermined temperature for running the chemical reactions within saidunit. In an embodiment, the plurality of temperature zones correspond tothe predetermined set of temperatures according to which the units arearranged.

In an embodiment where the apparatus and sample holder are configured toconduct a PCR reaction, the predetermined temperature zones correspondto different annealing temperatures at which multiple PCR reactions canbe run. In a further embodiment, the apparatus and sample holder arecapable of conducting a series of PCR reactions in order to amplifymost, if not all, of a genome. In this example, an entire genomerequires a range of annealing temperatures to achieve the desiredspecificity of a reaction. For example, these temperatures can begrouped into 2° C. temperature zones according to the annealingtemperatures of different nucleotide sequences. The units of a sampleholder can be addressed according to the temperature zone at which thereaction within the unit is to be run. In an embodiment, the apparatusreceives a sample holder that comprises a chip with units addressedaccording to six different annealing temperature zones corresponding tosix different temperature zones on the first heater of the apparatus.

In a preferable embodiment, the first and second heaters move betweenthe first and second orientations according to a protocol. The apparatuscan further comprise a motor for moving the first and second heatersbetween the first and second orientations. The heaters can be moved byany method as would be obvious to those skilled in the art.

The first orientation puts a heater in thermal contact with the sampleholder. In an embodiment, the sample holder is used to conduct a PCRreaction. The first heater has a plurality temperature zones, and can beused to provide the temperatures necessary for the annealing steps of aPCR reaction when the first heater is brought into thermal contact withthe sample holder. In an embodiment, the second heater provides atemperature necessary for elongation or denaturation of a nucleic acidduring a PCR reaction when the second heater is in the first orientationand thermal contact with a sample holder.

In an example of the apparatus and method of the invention, the secondheater is moved into the first orientation in thermal contact with thesample holder for a PCR reaction. The second heater delivers atemperature of around 95° C., in order to denature a nucleic acid in asample contained within the sample holder. After the denaturation step,the second heater is moved into the second orientation and the firstheater is moved into the first orientation in thermal contact with thesample holder. The first heater provides temperatures to the sampleholder for the annealing of a primer to a nucleic acid sequence of thesample. The process of conducting a denaturation step followed by anannealing step can be repeated or cycled until the desired amplificationproduct is achieved.

FIG. 12 demonstrates an example embodiment of a thermal cyclingapparatus 1200 of the invention. In this example, the apparatus 1200comprises a first heater 1210 with 6 different temperature zonescorresponding to 6 different addressable predetermined temperatures of achip 1250. The first heater 1210 can be moved in and out of thermalcontact with the chip 1250 as shown in the top view in FIG. 12. Thesecond heater 1220 provides a uniform temperature across the entireheater in order to heat each of the 6 different addressablepredetermined temperature areas of the chip 1250 to the sametemperature. For example, the second heater 1220 can provide uniformtemperatures for the denaturation and elongation steps of conducting PCRwith a chip of the invention, while the first heater 1210 can provide arange of annealing temperatures to increase the specificity ofconducting a large number of reactions. An imaging source 1260 foranalyzing the many reactions can be positioned on the opposite side ofthe chip from the first orientation of the heaters.

FIG. 13 demonstrates a side view of the first heater 1210 from theexample in FIG. 12 when the heater 1210 is in the first orientation inthermal contact with a chip with addressable units 1250. For example,each temperature zone 1212, 1214 of the heater can be provided by adifferent thermoelectric heating element 1216, 1218 and some examplespecifications are shown in FIG. 13.

In an embodiment, the first heater can provide a temperature gradient.Examples of types of heaters for the first and second heaters include,but are not limited to, a resistive heater and a thermoelectric heater.

In a further embodiment, the apparatus comprises a heat sink in thermalcontact with the first heater, the second heater, or both.

In order to monitor temperature, the apparatus can also comprise aplurality of temperature sensors. In an embodiment, the plurality oftemperature sensors have at least one temperature sensor assigned tomeasure the temperature of each temperature zone of the apparatus. Thetemperature sensor can be a thermocouple or any other sensor that areavailable in the art.

The heating element can be connected via electric leads to a powersource that provides voltage across the element and effects subsequentheating of the units. The heating element may also be coupled to atemperature sensor that monitors and regulates the temperature of theunit. The temperature sensor may control the temperature and hence thethermal profile of an array of units. Dividing the chip and/or firstheater into various temperature zones provides additional flexibilityfor parallel performance of chemical reactions that require differentthermal cycling profiles. Alternatively, the temperature sensor can becoupled to individual unit or zone so that the temperature of each unitor zone can be independently controlled. The temperature sensor may beincluded as a detachable unit located adjacent to or at the base of theunit. It can also be integrated into the interior or the exteriorsurface of the unit. Furthermore, the temperature sensor can befabricated as an integral part of the heating element.

A temperature profile provided by a thermal cycling apparatus of theinvention is demonstrated in FIG. 14. In this example, the 6 differenttemperature zones of the first heater are represented by separate lowertemperatures than the 95° C. uniform temperature provided by the secondheater. For example, when amplifying an entire human genome with thechips, systems, and apparatuses of the invention, 40 cycles can beperformed in less than about 15 minutes, 10 minutes or even 5 minutes inorder to amplify every gene (about 30,000) of the genome.

The body of the apparatus for providing cycling at least two temperaturelevels is configured to receive a sample holder. The sample holder canbe held in place within the body by a variety of means, including vacuumforce. In an embodiment, the body comprises a vacuum chuck for holdingthe sample holder firmly in place allowing for a heater to be placedinto thermal contact with the sample holder. Clamps, pins, adhesives,slots, or any other method of securing can be incorporated into the bodyas configured to receive the sample holder as would be obvious to thoseskilled in the art.

The apparatus can also further comprise sensors to determine theposition of the first and second orientations of a heating element inrespect to the sample holder.

In another aspect of the invention, an apparatus for conducting achemical reaction involving cycling at least two temperature levelscomprises: a body configured to receive a chip comprising a plurality ofnanowells for containing the chemical reaction; and a first heaterproviding a first temperature and a second heater providing a secondtemperature, wherein the first heater and the second heater areconfigured to be movable between a first and a second orientation, andwherein the first orientation places the heater in thermal contact withthe sample holder and the second orientation does not place the heaterin thermal contact with the sample holder.

In an embodiment, the plurality of nanowells are addressable, whereinthe nanowells are arranged according to a predetermined set oftemperatures, such that at least one of the nanowells is addressed toindicate the predetermined temperature for running the chemical reactionwithin said nanowell.

In another embodiment, the first heater comprises a plurality oftemperature zones. The plurality of temperature zones can correspond tothe predetermined set of temperatures according to which the units arearrayed.

The first and second heaters can move between the first and secondorientations according to a protocol. In an embodiment, the apparatuscan further comprise a motor for moving the first and second heatersbetween the first and second orientations.

In an embodiment, the first heater can provide a temperature gradient.Examples of types of heaters for the first and second heaters include,but are not limited to, a resistive heater and a thermoelectric heater.

In a further embodiment, the apparatus comprises a heat sink in thermalcontact with the first heater, the second heater, or both.

In order to monitor temperature, the apparatus can also comprise aplurality of temperature sensors. In an embodiment, the plurality oftemperature sensors have at least one temperature sensor assigned tomeasure the temperature of each temperature zone of the apparatus.

An exemplary apparatus and system 1500 of the invention is illustratedin FIG. 15. A chip 1510 can be loaded into an apparatus for conducting achemical reaction involving cycling at least two temperature levels. Thechip 1510 can be set in place by any method capable of holding them inplace, such as a vacuum or a clip. The chip can also be placed withinthe apparatus by sliding the chip into place against a stop or a wall,which aligns the chip in a proper position. The chip can be placed on asample holder 1520 that is capable of moving up and down. The sampleholder can be configured to couple to a heater 1530 of the apparatus1500.

In an embodiment, the chip 1510 and/or wells of the chip are sealed by athin plastic cover 1512, e.g., a standard PCR tape for covering a chipor a nanowell plate. The PCR tape can be made of a transparent material,such as polyethylene, and can be removable and sometimes can bereplaceable. Transparent oil can also be used to cover the wells and/orsurface of the chip 1510. In an embodiment, the chip 1510 and/or wellsare covered by oil and a cover.

In an embodiment, the cover 1512 covering the chip 1510 is not removedbefore placing the chip into a thermal cycling apparatus of theinvention. After the chip is in place within the apparatus, the heater1530 can provide a force to the chip 1510 that brings the chip 1510 intooptical or thermal contact with a top cover slide 1540. The top coverslide 1540 can be made of a transparent material or any material thatallows optics to view a reaction of the chip. Materials that can be usedas the top cover slide include, but are not limited to, glass, silica,silicon, and polymers or plastics as would be obvious to those skilledin the art.

The top cover slide 1540 may also comprise a heater 1542, such as anindium tin oxide (ITO) heater, that can heat the top of the chip 1510 orthe cover of the chip 1512 and/or wells. A heater 1542 of the top coverslide can be used to balance the temperature at the surface of the chipsuch that condensation does not occur at the surface or opening of aunit on the chip. For example, when a PCR reaction is run, the liquidcomponents of the reagents and/or sample may heat to a point where theycondense on the surface or cover of the reaction unit, or reaction well.Condensation can interfere with an optical system used to monitor thereaction within the unit. In an embodiment of the invention, asdemonstrated in FIG. 15, the heater 1530 of the apparatus can provide aforce that brings the chip 1510 into thermal contact with a top coverslide 1540 comprising a heater 1542, which can balance the temperatureat the surface of the chip 1510 to prevent condensation. Also shown inFIG. 15, the top cover slide 1540 may be connected to a bridge 1550 ofthe apparatus by a pair of springs or compressive devices 1552. Thesprings or other devices 1552 can relieve some of the pressure on thetop cover slide 1540 from the force of the heater 1530 against the chip1510, making the apparatus and system more robust. Any method of ordevice for pressure damping may be used to couple the top cover slide toa bridge of the apparatus.

In the exemplary system and apparatus in FIG. 15, a chip 1510 can beunloaded or loaded through the side of the thermal cycling apparatus1500. A heater 1530 of the apparatus can then be moved into anorientation in thermal contact with the chip 1510 and also provide aforce that brings the chip into thermal contact with a top cover slide1540. In FIG. 15, the chip has a plastic cover 1512 covering the wells.Also in FIG. 15, the top cover slide 1540 comprises fused SiO₂/Quartzmaterial and also comprises an ITO heater 1542. The ITO heater 1542 isoperated by electric leads 1544 connected to the ITO heater as shown inFIG. 15. The top cover slide 1540 is then connected to a bridge 1550 ofthe apparatus by a compressive spring 1552 that provides stress reliefwithin the system. The spring 1552 can also serve to improve the thermalcontact of the chip 1510 to both a heater 1530 of the apparatus and aheater of the top cover slide 1540.

In practice, controlling a heating element and hence the temperature ofthe reaction sample, can be effected by processing a predeterminedalgorithm stored on a computer readable medium operatively linked to theheating element. The movement of a heating element can also becontrolled by a protocol or algorithm, which can be provided by acomputer or stored on a computer readable medium. In other aspects, thecontrolling step may involve processing temperature or movement sensorsignals retrieved from a temperature sensor element that is operativelylinked to a unit of a sample holder or chip based on protocols stored ona computer readable medium. This can be achieved by employingconventional electronics components for temperature control that mayprocess either analog or digital signals. Preferably, the electronicscomponents are run on a feedback control circuitry. They can control thetemperature of one unit, but more often the temperature of multipleunits that collectively form one temperature zone or the temperature ofthe zone itself. In certain embodiments, the temperatures of thedifferent zones are separately controlled. The thermal cycling profileand duration will depend on the particular application in which thesubject chip is to be employed.

Systems

The subject chips can be provided with an optical system capable ofdetecting and/or monitoring the results or the progress of chemicalreactions taking place in the chips. Such optical system achieves thesefunctions by first optically exciting the reactants, followed bycollecting and analyzing the optical signals from the reactants of thechip. The optical system applicable for the present invention comprisesthree elements, namely the optical excitation element, the opticaltransmission element, and the photon-sensing element. The optical systemmay also comprise, optionally, an optical selection element.

FIG. 16 is a representative block diagram showing a representativeexample of the instrumentation in an experimental setup. FIG. 16 shows acomputer system (or digital device) 1600 connected to a laser 1610 as arepresentative example of an optical excitation element. The opticalexcitation element acts as the source of excitation beams used tooptically excite the reactants contained in the nanowells. This elementencompasses a wide range of optical sources that, generate light beamsof different wavelengths, intensities and/or coherent properties.Representative examples of such optical excitation sources include, butare not limited to, lasers, light-emitting diodes (LED), ultra-violetlight bulbs, and/or white light sources.

The optical transmission element used in the present invention servestwo functions. First, it collects and/or directs the optical excitationsources to the reactants inside the nanowells of the chips. Second, ittransmits and/or directs the optical signals emitted from the reactantsinside the nanowells of the chips to the photon-sensing element. Theoptical transmission element suitable for use in the present inventionencompasses a variety of optical devices that channel light from onelocation point to another. Non-limiting examples of such opticaltransmission devices include optical fibers, optical multiplexers (MUX)and de-multiplexers (DE-MUX), diffraction gratings, arrayed waveguidegratings (AWG), optical switches, mirrors, lenses, collimators, and anyother devices that guide the transmission of light through properrefractive indices and geometries.

The photon-sensing element analyzes the spectra of the optical signalscoming from the reactants inside the nanowells. Suitable photon-sensingelement can detect the intensity, of an optical signal at a givenwavelength, and preferably can simultaneously measure the intensities ofoptical signals across a range of wavelengths. Preferably the elementmay also provide spectrum data analyses to show the spectrum peakwavelength, spectrum peak width, and background spectrum noisemeasurements. Representative examples of suitable photon-sensing elementfor the present invention are avalanche photo diodes (APD),charge-coupled devices (CCD), electron-multiplying charge-coupled device(EMCCD), photo-multiplier tubes. (PMT), photo-multiplier arrays, gatesensitive FET's, nano-tube FET's, and P-I-N diode. As used herein, CCDincludes conventional CCD, electron-multiplying charge-coupled device(EMCCD) and other forms of intensified CCD.

While the subject optical systems can be assembled using manycombinations of the various elements, a useful assembly for analyzingthe spectra of the excited reactants comprises an optical transmissionelement and a photon-sensing element. Such assembly is also referred toherein as “spectrum analyzer”.

Where desired, the optical system of the present invention can includean optical selection element. This element selects and/or refines theoptical properties of the excitation beams before they reach thereactants contained in the nanowells. The optical selection element canalso be employed to select and/or refine the optical signals coming fromthe reactants in the nanowells before the signals reach thephoton-sensing element. Suitable optical selection element can selectand modify a wide range of optical properties, including but not limitedto, polarization, optical intensities, wavelengths, phase differencesamong multiple optical beams, time delay among multiple optical beams.Representative examples of such optical selection elements arepolarization filters, optical attenuators, wavelength filters (low-pass,band-pass or high-pass), wave-plates and delay lines.

The aforementioned optical elements can adopt a variety ofconfigurations. They can form integral parts of the subject chips orremain as separate units. All of these elements are commerciallyavailable. Accordingly, in one embodiment, the present inventionprovides a chip in which the optical transmission and photon-sensingelements are fabricated into the chip substrate. In one aspect, thephoton-sensing element is integrated into each nanowell on the chip thatis to be monitored. In another aspect, more than one type ofphoton-sensing element is integrated into the nanowell to enhance thedetection capability or efficiency. In another aspect, thephoton-sensing element can be fabricated along the side or at the baseof the nanowell, or as part of the cover of the nanowell. Photon-sensingelements suitable for such configuration include but are not limited toavalanche photo diode, charge coupled devices (including conventionalCCD, electron-multiplying charge-coupled device (EMCCD) and other formsof intensified CCD), gate sensitive FET's, nano-tube FET's, P-I-N diode.Avalanche photo diode is particularly preferred because it permitsdetections of a single photon by amplifying the signal through anavalanche process of electron transfer. These elements together with thesupporting circuitry can be fabricated as part of the subject chipsusing standard IC processing techniques described herein or known in theart.

In another embodiment, the present invention provides an apparatus inwhich the chip and the optical systems remain as separate units. Oneaspect of this embodiment encompasses an apparatus for conducting achemical or biological reaction requiring cycling at least twotemperature levels over a multiple-cycle period. The apparatus comprisesa chip of the present invention, and an optical system that isoperatively coupled to the chip and that detects an optical signalcoming from the nanowell. Preferably, the optical signals detected arerelated to the amount of product of the chemical reaction taking placein the nanowell.

FIG. 16 illustrates an exemplary optical system of this aspect. In anexemplary embodiment, this system includes an optical transmissionelement, such as a tunable laser 1610, or Xenon lamp, controlled by thecomputer or other digital setup 1600. The laser is then focused furtherto provide uniform distribution across all the nanowells using a Powelllens 1620, a telescope 1630, and/or a line focused laser 1640. Theoptical signals coming from the nanowells on the chip are collimated bya lens 1690, such as a tube lens, and are passed through a tunablefilter 1690, either a low-pass, high-pass, or notch-filter, to acharge-coupled device (CCD) 1615 for spectrum analysis. This particularembodiment offers a low cost solution for monitoring the progress and/orresults of chemical reactions taking place in nanowells fabricated on achip.

In a further embodiment the optical transmission element is moveablebeing placed on a X-Y stage, as seen in Option 1 of FIG. 16. In analternative embodiment, the chip is placed on an X-Y stage as seen inOption 2 of FIG. 16.

In a further embodiment feedback control or self learning is provided toachieve optimized chemical reactions at specific nanowells of thesubstrate. For example, using a fixed position substrate including anarray of nanowells in conjunction with a moveable optical transmissionelement and photon-sensing element, after heating (or providing a seriesof heating and/or cooling steps) with one or more optical transmissionunits, the photon-sensing element can detect the outcome of a desiredchemical reaction in a specific nanowell. Upon analysis of each chemicalreaction in a given nanowell based on the detection step, suitablecorrections (for example, raising or lowering reaction temperatures ordurations of reaction cycles) can be implemented in subsequent cyclerounds of heating and/or cooling to optimize the chemical reaction asneeded. This process can be repeated using repeated passes of thetransmission element and sensing element over the various nanowelllocations until the desired outcome of a chemical reaction is detectedin each nanowell. In this way a given nanowell chemical reaction can besequentially monitored and manipulated to provide an optimized chemicalreaction for the given nanowell. Different conditions may be required atdifferent nanowell locations based on the unique properties of theindividual reactions (for example, where different primers and templatesare used in a PCR reaction). Thus, the apparatus of the presentinvention can be used in conjunction with a feed back loop processdirected to each nanowell location, to optimize the chemical reaction atevery location on a substrate.

In an exemplary embodiment, a Chungara series CCD camera can be used.The Chungara CCD camera can be used for the most demanding applicationsin low noise and long exposure imaging in areas such as astronomy andspectroscopy. The Chungara CCD controller is able to read a wide rangeof CCDs, because it is based on modularity and common hardwarearchitecture. For instance, example CCDs that can be used range from the1536×1024 Kodak KAF-1602 CCD to the 4096×4096 Kodak KAF-16801 CCD. Thecamera can be linked to a host computer thru an ethernet link orwireless connection allowing a large distance between the computer andthe CCD camera.

In another embodiment, the present invention provides a system in whichthe chip, apparatus and the optical systems are separate units. In oneembodiment, the apparatus, system, chip of the present invention, athermal cycling apparatus or heating element of the invention, and anoptical system that is operatively coupled to the chip and that detectsan optical signal coming from an addressable unit of the chip.Preferably, the optical signals detected are related to the amount ofproduct of the chemical reaction taking place in the unit.

In another preferable embodiment, the amplified nucleic acids in thesubject chips are detected by the subject optical systems operativelycoupled to the chips. The optical systems are capable of transmittingappropriate excitation beams to the reactants in the amplificationreactions, collecting and analyzing the emitted optical signals from thereactants. Preferably, the optical signals detected are indicative ofthe amount of amplified nucleic acid in the amplification reaction overa multiple-cycle period. In certain aspects, the optical systemtransmits excitation beams into the wells containing the reactionsamples at a plurality of times during the amplification, and monitorsthe optical signals coming from the nanowells at each of the pluralityof times. By analyzing the relative intensities of the optical signals,preferably over a multiple-cycle period, one can monitor quantitativelythe progression of the amplification reaction. Typically, the opticalsignals being monitored are luminescent signals. Detecting and/ormonitoring the amplification products can be performed without openingthe nanowell once the amplification is initiated.

FIG. 17 demonstrates an example system 1700 of the invention with athermal cycling apparatus 1710 with a first 1712 and second heater 1712that can be moved between a first and second orientation, and a chip1720 with an addressable array of units based on predeterminedtemperatures. In this example, the optical system 1730 operativelycoupled to the chip 1720 and apparatus 1710 is a Chungara CCD camera1732. As shown by the side view in FIG. 17, the heaters 1712, 1714 canmove independently and brought into thermal contact with a chip 1720 ofthe invention to provide rapid thermal cycling of a large number ofchemical reactions, such as PCR amplification reaction of an entiregenome.

FIG. 18 demonstrates an example system 1800 of the invention comprisingan optical system 1810, a heating apparatus 1820, and a chip 1830 forconducting a chemical reaction. The heating apparatus 1820 of the systemmay be a thermal cycling device for conducting a PCR reaction. As shownin FIG. 18, the heating apparatus can comprise a denature block 1822 andan anneal block 1824 for conducting PCR. The heating apparatus 1820 isalso movable both horizontally and vertically as indicated by the arrowsin FIG. 18. A chip 1830 is inserted on a sample holder 1840 that canmove vertically. The chip 1830 and/or nanowells of the chip may or maynot be covered by a plastic cover 1832. The heater 1820 can force thesample holder 1840 into thermal contact with a top cover slide 1850comprising a thin film ITO heater 1852. The top cover slide 1850 isconnected, to the system 1800 by a compressive spring 1854 to permitbetter thermal contact between the sample holder 1840 and heaters 1820,1852 of the system. The system 800 may also comprise a dead stop 1856 toprevent the top cover slide 1850 from compressing into the opticalsystem 1810.

In FIG. 18, the optical system 1810 comprises a CCD camera 1812, anexcitation light source 1814, optics 1816, and an optical filter 1818.The system may comprise a plurality of optical systems. In anotherembodiment, the system comprises a plurality of CCD camera, excitationlight sources, optics, or optical filters. The optical system 1810 canfunction to receive information from a chemical reaction that occurs inthe chip 1830 when the chip 1830 is in thermal contact with the heatingapparatus 1820.

FIG. 19 is a block diagram showing a representative example logic devicethrough which reviewing or analyzing data relating to the presentinvention can be achieved. Such data can be in relation to a genotype, agenetic make up or a disease, disorder or condition in an individual.FIG. 19 shows a computer system (or digital device) 1900 connected to anapparatus 1920 for use with an apparatus 1924 to, for example, produce aresult. The computer system 1900 may be understood as a logicalapparatus that can read instructions from media 1911 and/or network port1905, which can optionally be connected, to server 1909 having fixedmedia 1912. The system shown in FIG. 19 includes CPU 1901, disk drives1903, optional input devices such as keyboard 1915 and/or mouse 1916 andoptional monitor 1907. Data communication can be achieved through theindicated communication medium to a server 1909 at a local or a remotelocation. The communication medium can include any means of transmittingand/or receiving data. For example, the communication medium can be anetwork connection, a wireless connection or an internet connection.Such a connection provide for communication over the World Wide Web. Itis envisioned that data relating to the present invention can betransmitted over such networks or connections for reception and/orreview by a party 1922. The receiving party or user 1922 can be apatient, a health care provider or a health care manager. In oneembodiment, a computer-readable medium includes a medium suitable fortransmission of a result of an analysis of a biological sample. Themedium can include a result regarding a genotype, a genetic make up or adisease condition or state of a subject, wherein such a result isderived using the methods described herein.

Uses of the Present Invention

The subject chips and apparatuses for thermal cycling have a widevariety of uses in chemical and biological applications wherecontrollable temperatures are desired. The methods, chips, andapparatuses of this invention are preferably performed with equipmentwhich aids in coupling one or more steps of the process, includinghandling of the chips, thermal cycling, and imaging. Accordingly, thepresent invention provides systems for simultaneously determining thegenetic expression profile in a biological sample obtained from anindividual member of a species relative to a standard genome for saidspecies.

In one embodiment, the invention can be used to vary and/or maintaintemperature of a reaction sample. Varying and/or maintaining temperatureof a reaction sample are required in a wide range of circumstancesincluding but not limited to discerning protein-protein interaction,examining DNA or RNA hybridization, and performing enzymatic reaction.The method involves placing the reaction sample into a nanowellfabricated in a chip that is in thermal contact with a heating element,and applying a voltage to the heating element.

In another embodiment, the subject chips apparatuses for thermal cyclingare used for conducting a chemical reaction that involves a plurality ofreaction samples and requires cycling at least two temperature levels.The process involves (a) providing a chip comprising an array of unitsas described herein; (b) placing the plurality of reaction samples intothe units of the chip; and (c) controlling the heating element, toeffect cycling at least two temperature levels.

Practicing the subject method generally proceeds with placing thereaction sample into a nanowell of the subject chip that is in thermalcontact with a heating element. Where desired, the reaction sample canbe applied by a dispensing system operatively coupled to the subjectchip. A variety of dispensing instruments, ranging from manuallyoperated pipettes to automated robot systems are available in the art.Preferred dispensing instruments include a piezo-electricnano-dispenser.

The subject chips and apparatuses are particularly suited for conductingquantitative nucleic acid amplification. Accordingly, the presentinvention provides a method for monitoring the formation of a nucleicacid amplification reaction product, preferably in real time. In certainpreferred embodiments, the amplified nucleic acids contained aredirectly monitored by the photon-sensing elements integrated into thechips. The photon-sensing element registers the intensities of theoptical signals that are reflective of the amount of the amplifiednucleic acids at any time being examined during the amplificationreaction. The optical signals may be any kind of luminescent signalsemitted upon exciting the labeled reactants with appropriate incidentbeams.

The subject methods of amplifying and detecting a target nucleic acidhave broad spectrum of utility in, for example drug screening, diseasediagnosis, phylogenetic classification, genotyping individuals, parentaland forensic identification.

In an embodiment, a system, chip, apparatus, or method of the inventioncan be used to discover therapeutically-relevant biomarkers. Forexample, the invention could be used to identify biomarkers for chronicobstructive pulmonary disease (COPD) and lung cancer.

At a more fundamental level, amplification and detection of the targetnucleic acids may be used in identification and quantification ofdifferential gene expression between diseased and normal tissues, amongdifferent types of tissues and cells, amongst cells at differentdevelopmental stages or at different cell-cycle points, and amongstcells that are subjected to various environmental stimuli or lead drugs.

In various configurations of the present invention, a method is disclosefor supplying to a consumer assays useful in obtaining structuralgenomic information, such as the presence or absence of one or moresingle nucleotide polymorphisms (SNPs), and functional genomicinformation, such as the expression or amount of expression of one ormore genes. As such, the assays can be configured to detect the presenceor expression of genetic material in a biological sample. The methodincludes providing a user interface configured for receiving orders forstock assays, providing a user interface configured for receivingrequests for design of custom assays and for ordering said assays, anddelivering to the consumer at least one custom or stock assay inresponse to an order for the one custom or stock assay placed by theconsumer. In certain other aspects, the present invention includes asystem, apparatus, chip, and methods for constructing a system forproviding to a consumer assays configured to detect presence orexpression of genetic material. In an embodiment, the chips can becustomized according to a user's needs.

The present invention provides devices and methods for containing andhandling small quantities of liquids, including methods and devices forperforming amplification reactions on liquid samples containingpolynucleotides. Embodiments of the present invention include chips forconducting a chemical reaction, including a thermocycled amplificationreaction of polynucleotide, in a liquid sample.

The subject chips and other devices find utility in many other chemicaland biological applications where controllable temperatures are desired.Such applications include a vast diversity of reactions such as redoxreactions, hydrolysis, phosphorylation, and polymerization. Additionalapplications are directed to discerning interactions involvingbiological molecules such as proteins, glycoproteins, nucleic acids, andlipids, as well as inorganic chemicals, or any combinations thereof. Thechemical reaction may also involve interactions between nucleic acidmolecules, between nucleic acid and protein, between protein and smallmolecules. The chemical reaction may take place outside a cell or insidea cell that is introduced into a nanowell of the subject chip.

Of particular significance is the application in detecting the presenceof a specific protein-protein interaction. Such application generallyemploys a proteinaceous probe and a target protein placed in a unit inthe subject chip.

In one aspect of this embodiment, the protein-protein interaction isbetween a target protein (for example an antigen) and an antibodyspecific for that target. In another aspect, the protein-proteininteraction is between a cell surface receptor and its correspondingligand. In yet another aspect, the protein-protein interaction involvesa cell surface receptor and an immunoliposome or an immunotoxin; inother aspects, the protein-protein interaction may involve a cytosolicprotein, a nuclear protein, a chaperon protein, or proteins anchored onother intracellular membranous structures.

The terms “membrane”, “cytosolic”, “nuclear” and “secreted” as appliedto cellular proteins specify the extracellular and/or subcellularlocation in which the cellular protein is mostly, predominantly, orpreferentially localized.

“Cell surface receptors” represent a subset of membrane proteins,capable of binding to their respective ligands. Cell surface receptorsare molecules anchored on or inserted into the cell plasma membrane.They constitute a large family of proteins, glycoproteins,polysaccharides and lipids, which serve not only as structuralconstituents of the plasma membrane, but also as regulatory elementsgoverning a variety of biological functions.

The reaction is typically performed by contacting the proteinaceousprobe with a target protein under conditions that will allow a complexto form between the probe and the target. The conditions such as thereaction temperature, the duration of the reaction, the bufferconditions and etc., will depend on the particular interaction that isbeing investigated. In general, it is preferable to perform thereactions under physiologically relevant temperature and bufferconditions. Physiologically relevant temperatures range fromapproximately room temperature to approximately 37° C. This can beachieved by adjusting the heating element of the subject chips.Typically, a physiological buffer contains a physiological concentrationof salt and at adjusted to a neutral pH ranging from about 6.5 to about7.8, and preferably from about 7.0 to about 7.5. A variety ofphysiological buffers is listed in Sambrook et al. (1989) supra andhence is not detailed herein.

The formation of the complex can be detected directly or indirectlyaccording standard procedures in the art or by methods describe herein.In the direct detection method, the probes are supplied with adetectable label and when a complex is formed, the probes emitted anoptical signal distinct from that of the unreacted probes. A desirablelabel generally does not interfere with target binding or the stabilityof the resulting target-probe complex. As described above, a widevariety of labels suitable for such application are known in the art,most of which are luminescent probes. The amount of probe-targetcomplexes formed during the binding reaction can be quantified bystandard quantitative assays, or the quantitative methods using theoptical systems described above.

The examples and other embodiments described herein are exemplary andare not intended to be limiting in describing the full scope ofapparatus, systems, compositions, materials, and methods of thisinvention. Equivalent changes, modifications, variations in specificembodiments, apparatus, systems, compositions, materials and methods maybe made within the scope of the present invention with substantiallysimilar results. Such changes, modifications or variations are not to beregarded as a departure from the spirit and scope of the invention.

1. An apparatus comprising at least one heating element, configured tobe in thermal contact with a chip said chip comprising a substrate andan array of nanowells, wherein the at least one heating element isconfigured to move relative to the chip.
 2. The apparatus of claim 1,wherein the at least one heating element is in thermal contact with thechip from above and below the chip, and wherein the heating element inthermal contact from below the chip is set at a temperature lower thanthe temperature of the heating element in thermal contact from above thechip.
 3. The apparatus of claim 1, wherein the chip comprises an uppersurface and a bottom surface and wherein a first series of nanowells isarranged along one orientation on the upper surface and a second seriesof nanowells is oriented perpendicular to the first series of nanowells.4. The apparatus claim 1, wherein the heating element is positionedabove or below a stationary chip comprising an array of nanowells. 5.The apparatus of claim 1, wherein the heating element is capable ofheating and cooling.
 6. The apparatus of claim 1, comprising a pluralityof heating elements corresponding to a plurality of temperature zones.7. The apparatus of claim 6, wherein the plurality of temperature zonesa range from about 52° C. to about 95° C.
 8. The apparatus of claim 7,wherein the plurality of temperature zones provides a temperaturegradient.
 9. The apparatus of claim 6, wherein at least one of thetemperature zones is set at a temperature ranging from about 52° C. toabout 65° C. and at least one other temperature zone is set at atemperature ranging from about 90° C. to about 95° C.
 10. The apparatusof claim 9, further comprising at least one other temperature zone setat temperature ranging from about 68° C. to about 72° C.
 11. Theapparatus of claim 1, wherein the at least one heating element isconfigured to provide an output comprising a spike waveform oftemperature over time.
 12. The apparatus of claim 1, wherein anindividual nanowell in said array has a dimension of about 250 μm inlength, about 250 μm in width, and a depth of about 525 μm, or less. 13.The apparatus of claim 1, wherein the chip is operatively coupled to anoptical system that detects optical signals.
 14. The apparatus of claim13, wherein the optical system comprises a plurality of opticaldetectors.
 15. The apparatus of claim 1, wherein the number of nanowellsis greater than about 30,000.
 16. The apparatus of claim 1, wherein thenanowells are configured to contain about 100 nl.
 17. A method ofconducting a chemical reaction comprising: providing a chip configuredto receive a reaction sample; providing at least one heating elementpositionable in relation to the chip to provide thermal contact betweenthe at least one heating element and the chip; and conducting thechemical reaction in the reaction sample by varying the temperature ofthe chip, wherein said varying the temperature is effected by moving theat least one heating element in relation to the chip such that theheating element is in thermal contact with the chip.
 18. The method ofclaim 17, wherein the chemical reaction is a nucleic acid amplificationreaction.
 19. The method of claim 17, wherein movement of the at leastone heating element is controlled by signals generated from atemperature sensor that is operatively linked to the chip.
 20. Themethod of claim 18, wherein the reaction sample is capable of producingan optical signal, and, wherein the chip is operatively coupled to anoptical system configured to detect optical signals emitted from thereaction sample.
 21. The method of claim 20, wherein the optical signalsare proportional to the amount of product of the chemical reaction. 22.The method of claim 21, wherein said varying the temperature is effectedby moving a plurality of heating elements, each of which is set at adifferent temperature.
 23. The method of claim 21, wherein at least oneheating element is set at a temperature ranging from about 52° C. toabout 65° C. and at least one other heating element is set at atemperature ranging from about 90° C. to about 95° C.
 24. A chip forrunning a reaction comprising an array of addressable units, each unitbeing configured for running a chemical reaction, wherein the array ofthe addressable units is configured to correspond to a predeterminedtemperature zone, and wherein an individual unit in said array isdimensioned to hold a chemical reaction mixture of less than about 1 μl.25. The chip of claim 24 comprising a plurality of arrays.
 26. The chipof claim 24 comprising a plurality of arrays, each of whichcorresponding to a different temperature zone.
 27. The chip of claim 24comprising a plurality of arrays wherein at least one of the arrays isset at an annealing temperature for supporting a nucleic acidamplification reaction and at least one other array is set at adenaturing temperature for supporting a nucleic acid amplificationreaction.
 28. The chip of claim 24, wherein the zone is addressed toindicate the predetermined temperature zones.
 29. The chip of claim 24,wherein the array of addressable units are configured to correspond tosix or more predetermined temperature zones.
 30. The chip of claim 24,wherein the chip is in thermal contact with a heating element.
 31. Anapparatus for conducting a chemical reaction requiring cycling at leasttwo temperature levels, comprising: (a) chip for running a reactioncomprising an array of addressable units, each unit being configured fora chemical reaction, wherein the array of the addressable units isconfigured to correspond to a predetermined temperature zone, andwherein an individual unit in said array is dimensioned to hold achemical reaction mixture of less than about 1 μl; and (b) a heatingelement in thermal contact with the chip.
 32. The apparatus of claim 24or claim 31, wherein the array of addressable units is greater thanabout 30,000.
 33. The apparatus of claim 31, further comprising (c) anoptical system operatively coupled to the chip, wherein the opticalsystem detects an optical signal coming from an addressedthermo-controllable unit.
 34. The apparatus of claim 33, wherein theoptical system comprises a plurality of optical detectors.
 35. Theapparatus of claim 31, further comprising a plurality of heatingelements.
 36. The apparatus of claim 35, wherein the plurality ofheating elements comprises six or more heating elements.
 37. Theapparatus of claim 24 or claim 31, wherein an individual unit within thearray comprises a nanowell for receiving and confining a sample, saidwell being sealed when filled with the sample.
 38. The apparatus ofclaim 24 or claim 31, wherein the chemical reaction is a nucleic acidamplification reaction.
 39. The apparatus of claim 38, wherein thepredetermined temperature of a unit is configured to yield at least 90%of homogeneous product from the chemical reaction.
 40. A method ofconducting a reaction that involves a plurality of reaction samples andrequires cycling at least two temperature levels, comprising: (a)providing a chip for running a reaction comprising an array ofaddressable units, each unit being configured for a chemical reaction,wherein the array of the addressable units is configured to correspondto a predetermined temperature zone, and wherein an individual unit insaid array is dimensioned to hold a chemical reaction mixture of lessthan about 1 μl; (b) placing the plurality of reaction samples into theunits of the chip according to the set of predetermined temperatures;and (c) controlling a heating element to effect cycling at least twotemperature levels.
 41. The method of claim 40, wherein an individualunit within the array of the chip comprises a nanowell for receiving andconfining a sample, said well being sealed when filled with the sample.42. The method of claim 40, wherein the chemical reaction is a nucleicacid amplification reaction.
 43. The method of claim 42, wherein thepredetermined temperature of a unit is configured to yield at least 90%of homogeneous product from the chemical reaction.
 44. An apparatus forconducting a chemical reaction involving cycling at least twotemperature levels, comprising: (a) a body configured to receive a chipcomprising a plurality of nanowells, for containing the chemicalreaction; and (b) a first heater providing a first temperature and asecond heater providing a second temperature; wherein the first heaterand the second heater are configured to be movable between a first and asecond orientation, and wherein the first orientation places the heaterin thermal contact with the sample holder and the second orientationdoes not place the heater in thermal contact with the sample holder. 45.The apparatus of claim 44, wherein the plurality of nanowells areaddressable, wherein the nanowells are arranged according to apredetermined set of temperatures such that at least one of thenanowells is addressed to indicate the predetermined temperature forrunning the chemical reaction within said nanowell.
 46. The apparatus ofclaim 44, wherein the plurality of nanowells comprises over about 30,000nanowells.
 47. The apparatus of claim 44, wherein the first heatercomprises a plurality of temperature zones.
 48. The apparatus of claim47, wherein the temperature zones comprise six or more temperaturezones.
 49. The apparatus of claim 48, wherein the plurality oftemperature zones correspond to the predetermined set of temperaturesaccording to which the thermo-controllable units are arrayed.
 50. Theapparatus of claim 44, wherein the first and second heaters move betweenthe first and second orientations according to a protocol.
 51. Theapparatus of claim 44 further comprising a motor for moving the firstand second heaters between the first and second orientations.
 52. Theapparatus of claim 44, wherein the first heater provides a temperaturegradient.
 53. The apparatus of claim 44, further comprising a heat sinkin thermal contact with the first heater.
 54. The apparatus of claim 44,further comprising a heat sink in thermal contact with the secondheater.
 55. The apparatus of claim 53 or claim 54, further comprising afan for removing heat from the heat sink.
 56. The apparatus of claim 44,further comprising a plurality of temperature sensors operably connectedto the chip.
 57. The apparatus of claim 56, wherein the plurality oftemperature sensors has at least one temperature sensor assigned tomeasure the temperature of each temperature zone.